Stimulation Of Cytotoxic T Lymphocytes Research Articles

The Role of Viral‐specific Immune Responses on the Outcome of Chronic Hepatitis B (HBV) Infection in Children: New Insights Into Immunopathology

The Role of Viral‐specific Immune Responses on the Outcome of Chronic Hepatitis B (HBV) Infection in Children: New Insights Into Immunopathology

Blood donor derived dendritic cells and cytotoxic T cells for specific fusion‐gene adoptive immunotherapy

Therapeutic immunological reagents tailored to individual patients have been shown to be a viable treatment strategy for some forms of leukaemia. This work investigates the possibility of using blood donations as a source of leukaemia-specific immune therapeutics. The acute promyelocytic cell line NB4 carrying the PML-RAR alpha fusion was used as a target for cytotoxic T lymphocytes (CTL) stimulated to recognize the fusion. Stimulation of CTL was by production of dendritic cells pulsed with plasmid vectors containing polymerase chain reaction (PCR)-generated sequences of PML-RAR alpha derived from NB4 cells. PCR primer design included a Kozak consensus sequence to allow correct translation of the nucleic acid into protein. Identification of specific cytotoxicity was by both Granzyme B ELISPOT and by (51)Cr-release assays. Specific CTL activity targeting NB4 cells can be generated from donor-derived peripheral blood mononuclear cells. However, individual donors appear to respond differently to the length of stimulatory sequence encoded in the vector. Use of an internal methionine in the PML gene, which also satisfies the Kozak rules, allows translation in vitro and, thus, might provide a suitable start site for stimulation using acute promyelocytic leukaemia-specific sequence. The work presented here suggests that blood donor derived dendritic cells can be used to stimulate leukaemia-specific CTL from the same donation ex vivo. This would enable the generation of patient-specific therapeutics from major histocompatibility (MHC)-matched allogeneic donors. However, different MHC-matched donors might vary in their response depending on the length of the antigenic sequence.

Concomitant Induction of CMVpp65-Specific CD4+ and CD8+ T Cells Using Dendritic Cells Transfected with mRNA Encoding an Invariant Chain-pp65 Fusion Protein.

Human cytomegalovirus (CMV) disease is increasingly recognized as a major cause of morbidity and mortality in post-stem cell transplant (SCT) recipients due to a lack of cellular immunity. Thus, novel therapies that offer the restoration of cellular immunity in SCT patients are highly desirable for clinical use. Cytotoxic T lymphocyte (CTL) responses against CMV represent a major effector arm of the immune system to control viremia. However, it is well established that the efficient induction and persistence of CTL responses in vivo requires the concomitant induction of antigen-specific CD4+ T helper cells. In this study, we sought to determine whether human dendritic cells (DC) transfected with mRNA encoding an invariant chain-CMVpp65 fusion protein (Ii-pp65) were capable of inducing concomitant CMV-specific CTL and CD4+ responses, thereby constituting a useful strategy for immunotherapy of CMV disease. We show that transfection of DC with Ii-pp65 mRNA leads to enhanced stimulation of CMV-specific CTL in vitro (Figure 1). Furthermore, DC expressing Ii-pp65 are potent inducers of primary CMV-specific CD4+ T cell responses as evidenced by ELISPOT analyses of primed CD4+CD45RA+ T cells (Figure 2). Lastly, efficient routing of Ii-pp65 into the MHC class II presentation pathway is demonstrated by confocal microscopy (data not shown). Based on these preclinical findings, we propose a clinical trial to administer DC, transfected with mRNA encoding a chimeric Ii-pp65, to post-SCT patients. Our primary goal will be to prevent CMV infection and reactivation by inducing strong immune reactivities against CMV. [Display omitted]

Generation of melanoma-specific cytotoxic T lymphocytes for allogeneic immunotherapy.

To exploit alloreactive T-cell responses known as the graft-versus-tumor effect, allogeneic hematopoietic stem cell transplantation is being explored as experimental therapy in selected solid tumors, including metastatic melanoma. However, donor T-cell responses are often delayed and associated with severe graft-versus-host disease. Posttransplant adoptive immunotherapy using tumor-specific cytotoxic T lymphocytes (CTL) of donor origin might provide immediate graft-versus-tumor effects but not graft-versus-host disease. Therefore, the aim of the current study was to define in vitro conditions for the expansion of allogeneic major histocompatibility complex-matched CTLs targeting melanoma-associated antigens (MAA). The CTLs were generated from peripheral blood mononuclear cells (PBMCs) of HLA-A*0201+ healthy donors by repetitive stimulations with HLA-A*0201-restricted MAA-derived peptides. Melanoma reactivity, as determined by lysis of peptide-pulsed T2 cells and HLA-A2+/Ag+ melanoma cells, was detected using in vitro expanded CTL targeting MAA peptides AAGIGILTV(MT(27-35)), IMDQVPFSV(G(209-2M)), and YMDGTMSQV(T(368-376)). In contrast, FLWGPRALV(MAGE3(271)-(279)) and VLPDVFIRCV(GnT-V(nt38-67)) induced peptide-specific recognition of T2 target cells only, whereas ITDQVPFSV(G(209-217)), KTWGQYWQV(G9(154)), MLLAVLYCL(T(1-9)), and tumor lysate could not induce specific CTLs. Specific cytolytic activity was accompanied by interferon-gamma secretion. Peptide-pulsed dendritic cells were required only for the initial stimulation of CTLs and could be substituted by PBMCs during restimulations. The median expansion rate of CTL was five to six times, regardless of whether dendritic cells or PBMCs were used after the initial stimulation. The results delineate the conditions for effective ex vivo expansion of melanoma-specific CTLs from PBMCs of healthy donors to be used as an adjunct in allogeneic cell therapy of metastatic melanoma.

Generating CTLs against the subdominant Epstein-Barr virus LMP1 antigen for the adoptive immunotherapy of EBV-associated malignancies

The Epstein-Barr virus (EBV)-encoded LMP1 protein is expressed in EBV-positive Hodgkin disease and is a potential target for cytotoxic T-lymphocyte (CTL) therapy. However, the LMP1-specific CTL frequency is low, and so far the generation of LMP1-specific CTLs has required T-cell cloning. The toxicity of LMP1 has prevented the use of dendritic cells (DCs) for CTL stimulation, and we reasoned that an inactive, nontoxic LMP1 mutant (DeltaLMP1) could be expressed in DCs and would enable the activation and expansion of polyclonal LMP1-specific CTLs. Recombinant adenoviral vectors expressing LMP1 or DeltaLMP1 were tested for their ability to transduce DCs. LMP1 expression was toxic within 48 hours whereas high levels of DeltaLMP1 expression were achieved with minimal toxicity. DeltaLMP1-expressing DCs were able to reactivate and expand LMP1-specific CTLs from 3 healthy EBV-seropositive donors. LMP1-specific T cells were detected by interferon-gamma (IFN-gamma) enzyme-linked immunospot assay (ELISPOT) assays using the HLA-A2-restricted LMP1 peptide, YLQQNWWTL (YLQ). YLQ-specific T cells were undetectable (less than 0.001%) in donor peripheral blood mononuclear cells (PBMCs); however, after stimulation the frequency increased to 0.5% to 3.8%. Lysis of autologous target cells by CTLs was dependent on the level of LMP1 expression. In contrast, the frequency of YLQ-specific CTLs in EBV-specific CTLs reactivated and expanded using lymphoblastoid cell lines was low and no LMP1-specific cytotoxic activity was observed. Thus, DeltaLMP1 expression in DCs is nontoxic and enables the generation of LMP1-specific CTLs for future adoptive immunotherapy protocols for patients with LMP1-positive malignancies such as EBV-positive Hodgkin disease. Targeting LMP1 in these malignancies may improve the efficacy of current adoptive immunotherapy approaches.

Presentation of native TROP-2 tumor antigens to human cytotoxic T lymphocytes by engineered antigen-presenting cells.

Professional antigen-presenting cells (APC), e.g. dendritic cells, express immuno-proteasome components and process proteins for MHC presentation differently from non-immune cells. Thus, they induce reactivities against sets of peptides that do not overlap with those generated by non-professional APC, i.e., tumor cells, and stimulate cytotoxic T lymphocytes (CTL) that may not recognize them. The goal of this work was to establish a system for antigen presentation and in vitro stimulation of human CTL using "tumor-cell-like" engineered APC. Murine fibroblasts were transfected with human HLA Class I alleles, together with the B7.1, ICAM-1 and germ-line TROP2 genes. The last encodes a cell surface glycoprotein widely expressed by human cancers. Unseparated peripheral blood mononuclear cells from HLA Class I-matched individuals were stimulated in vitro by the engineered APC. These efficiently induced the activation and proliferation of antigen-specific HLA-restricted CTL lines and clones. The Trop-2-specific CTL demonstrated high specific cytotoxicity against the appropriate transfected target cells. They also efficiently lysed MCF-7 human tumor cells expressing endogenous HLA-A2.1, Trop-2 together with ICAM-1. These results demonstrate that Trop-2 is a target molecule recognized by human CTL. Moreover, they demonstrate that non-immune engineered APC efficiently process and present native tumor-specific proteins in the context of human MHC Class I, and stimulate the growth and cytotoxicity of specific anti-tumor CTL.

The role of CD8(+) T cells in immune responses to colorectal cancer.

Cytotoxic T lymphocytes (CTL) are essential effectors of the cell-mediated immune response. The ability of CTL to specifically recognise and lyse malignant cells expressing the relevant surface antigens under optimal in vitro conditions justifies attempts to boost their number and activity through various forms of immunotherapy. Considering the high prevalence of colorectal cancer and poor survival rates for patients with advanced-stage disease, the development of new protocols based on CTL stimulation represents a genuine and promising treatment option. Significant advances in recombinant DNA technology and molecular biology have led to the identification of a number of tumour-associated antigens (TAA). These have served as vaccine constituents and/or stimuli for obtaining CTL used for adoptive immunotherapy after in vitro stimulation and expansion. The present review describes the properties and functions of CTL as effectors of the immune response against tumours, and summarises the known TAA recognised by CTL and the current status of CTL-related immunotherapeutic interventions in colorectal cancer patients.

Induction of simian immunodeficiency virus (SIV)-specific CTL in rhesus macaques by vaccination with modified vaccinia virus Ankara expressing SIV transgenes: influence of pre-existing anti-vector immunity

A major aim in AIDS vaccine development is the definition of strategies to stimulate strong and durable cytotoxic T lymphocyte (CTL) responses. Here we report that simian immunodeficiency virus (SIV)-specific CTL developed in 4/4 macaques following a single intramuscular injection of modified vaccinia virus Ankara (MVA) constructs expressing both structural and regulatory/accessory genes of SIV. In two animals Nef-specific responses persisted, but other responses diminished and new responses were not revealed, following further vaccination. Vaccination of another two macaques, expressing Mamu A*01 MHC class I, with MVA constructs containing nef and gag-pol under the control of the moderate strength natural vaccinia virus early/late promoter P7.5, again induced an early Nef-specific response, whereas responses to Gag remained undetectable. Anti-vector immunity induced by this immunization was shown to prevent the efficient stimulation of CTL directed to the cognate Gag epitope, p11C C-M, following vaccination with another MVA construct expressing SIV Gag-Pol under a strong synthetic vaccinia virus-specific promoter. In contrast, vaccination of a previously unexposed animal resulted in a SIV-specific CTL response widely disseminated in lymphoid tissues including lymph nodes associated with the rectal and genital routes of SIV entry. Thus, despite the highly attenuated nature of MVA, repeated immunization may elicit sufficient anti-vector immunity to limit the effectiveness of later vaccination.

Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells

AbstractDesigning effective strategies to load human dendritic cells (DCs) with tumor antigens is a challenging approach for DC-based tumor vaccines. Here, a cytoplasmic expression system based on mRNA electroporation to efficiently introduce tumor antigens into DCs is described. Preliminary experiments in K562 cells using an enhanced green fluorescent protein (EGFP) reporter gene revealed that mRNA electroporation as compared with plasmid DNA electroporation showed a markedly improved transfection efficiency (89% versus 40% EGFP+ cells, respectively) and induced a strikingly lower cell toxicity (15% death rate with mRNA versus 51% with plasmid DNA). Next, mRNA electroporation was applied for nonviral transfection of different types of human DCs, including monocyte-derived DCs (Mo-DCs), CD34+ progenitor-derived DCs (34-DCs) and Langerhans cells (34-LCs). High-level transgene expression by mRNA electroporation was obtained in more than 50% of all DC types. mRNA-electroporated DCs retained their phenotype and maturational potential. Importantly, DCs electroporated with mRNA-encoding Melan-A strongly activated a Melan-A–specific cytotoxic T lymphocyte (CTL) clone in an HLA-restricted manner and were superior to mRNA-lipofected or -pulsed DCs. Optimal stimulation of the CTL occurred when Mo-DCs underwent maturation following mRNA transfection. Strikingly, a nonspecific stimulation of CTL was observed when DCs were transfected with plasmid DNA. The data clearly demonstrate that Mo-DCs electroporated with mRNA efficiently present functional antigenic peptides to cytotoxic T cells. Therefore, electroporation of mRNA-encoding tumor antigens is a powerful technique to charge human dendritic cells with tumor antigens and could serve applications in future DC-based tumor vaccines.

Two intermediate-avidity cytotoxic T lymphocyte clones with a disparity between functional avidity and MHC tetramer staining.

The efficacy of cytotoxic T lymphocytes (CTL) has been shown to be highly dependent upon their functional avidity (the sensitivity of their cellular response to MHC-peptide complexes). To examine this relationship, we employed target cell lysis as a quantitative measure and established a set of four CTL clones that exhibited a range of functional avidities spanning more than three orders of magnitude. Within this set, clones displayed a linear correlation between functional avidity and the TCR down-regulation that occurred in response to increasing antigen density. Staining intensity of MHC-peptide tetramer, however, correlated only with the very highest and very lowest avidity clones; the two intermediate-avidity clones showed an inverse relationship between tetramer staining and functional avidity. Compensation for differences in surface levels of TCR improved the correlation, but failed to fully account for this discrepancy. Comparison of TCR signals generated by stimulation of CTL with substrate-bound soluble MHC-peptide or antigen-presenting cells suggested that internal TCR signaling efficiency accounts for at least a portion of the observed functional avidity and suggests the need for caution in directly relating tetramer staining to avidity.

Development of semisynthetic triterpenoid saponin derivatives with immune stimulating activity

Aldehyde-containing triterpene saponins have adjuvant properties, but only those from Quillaja saponaria Molina stimulate the production of cytotoxic T lymphocytes (CTL) against exogenous antigens. Quillaja saponins have two normonoterpene ester moieties, linked linearly to their fucosyl residue, that play a critical role in the stimulation of CTL. These ester moieties are also responsible for these saponins’ instability and toxicity. Based on the structure–activity relationships for the different groups of Q. saponaria saponins, new semi-synthetic analogs were developed that have the adjuvanticity of quillaja saponins, yet with less toxicity and greater stability in aqueous solutions. The quillaja saponin analogs were prepared by replacing their hydrolytically unstable ester groups with another lipophilic chain linked by a stable amide bond on these saponins’ glucuronic acid residue. One of these analogs, GPI-0100, is a dodecylamide saponin derivative that stimulates an antibody isotype profile that corresponds to a Th1 type immune response, as well as CTL production against exogenous antigens.

Induction of human cytotoxic T lymphocytes by artificial antigen-presenting cells.

The adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) is a promising therapeutic approach for a number of diseases. To overcome the difficulty in generating specific CTLs, we established stable artificial antigen-presenting cells (AAPCs) that can be used to stimulate T cells of any patient of a given human leukocyte antigen (HLA) type. Mouse fibroblasts were retrovirally transduced with a single HLA-peptide complex along with the human accessory molecules B7.1, ICAM-1, and LFA-3. These AAPCs consistently elicit strong stimulation and expansion of HLA-restricted CTLs. Owing to the high efficiency of retrovirus-mediated gene transfer, stable AAPCs can be readily engineered for any HLA molecule and any specific peptide.

Identification of subdominant cytotoxic T lymphocyte epitopes encoded by autologous HIV type 1 sequences, using dendritic cell stimulation and computer-driven algorithm.

Conventional analysis of the cytotoxic T lymphocyte (CTL) response to HIV-1 may underestimate the true breadth of CTL epitopes recognized. This underestimation could be due to several reasons, including (1) the use of laboratory-adapted stains of HIV or consensus sequences, which would lead to the identification of only highly conserved epitopes, (2) the use of EBV-transformed B cells (B-LCLs) and vaccinia virus constructs in standard assays that may obscure low level CTL responses due to high EBV or vaccinia reactivity, and (3) relatively insensitive assays wherein PBMCs instead of professional APCs are used to stimulate CTL responses. To address these problems, we first identified an immunodominant HLA-B7-restricted CTL epitope, by standard cloning methods, in a long-term nonprogressor (LTNP). To determine whether the patient had CTLs specific for autologous viral sequences other than the dominant epitope, proviral DNA was cloned and sequenced. A matrix-based epitope algorithm (EpiMatrix) was used to identify the top 2% of peptides from the viral sequences with the highest likelihood of binding to HLA-B7. These 55 peptides were synthesized and tested for HLA-B7 binding in a T2/B7 cell line; 10 peptides were able to stabilize HLA-B7 on the cell surface. By using peptide-pulsed autologous dendritic cells as a more sensitive method of CTL stimulation, we found three additional subdominant CTL epitopes.

Thioamides: synthesis, stability, and immunological activities of thioanalogues of Imreg. Preparation of new thioacylating agents using fluorobenzimidazolone derivatives.

Imreg (Tyr1-Gly2-Gly3) is a well-known immunostimulant. However, it possesses a short half-life. Stabilized analogues of Imreg were prepared by a regioselective insertion in which peptide bonds at position 1,2 or 2,3 were replaced by thioamide linkages. This was achieved by using new thioacylating agents based on thioacyl-fluoro-N-benzimidazolone. The synthesis and properties of these reagents are described herein. This peptide modification enhanced significantly the half-life of the thioanalogues relative to Imreg in blood. The thioanalogues and Imreg were tested in vitro in T and B cell proliferation assays and for their ability to stimulate cytotoxic T-lymphocytes (CTLs). Only thiotyrosyl glycyl glycine 11 displayed some activity as evidenced by a weak stimulation of CTLs. On the basis of this activity and the increased stability, an in vivo immunological evaluation was undertaken. Immunophenotyping of 11 revealed a significant increase in activated CTL and NK cell populations in the spleen. This expansion was also accompanied by a significant stimulation of NK cells and the B cell proliferative response. Thioanalogues of Imreg were generally nontoxic, as exemplified by 11. The latter is a promising immunostimulant which may be targeted for cancer and viral infections, where CTLs and NK cells play an important role, or as a vaccine adjuvant where stimulation of antibody-producing B cells is important.

Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs.

CD8+ cytotoxic T lymphocytes (CTLs) mediate resistance to infectious agents and tumours. Classically, CTLs recognize antigens that are localized in the cytoplasm of target cells, processed and presented as peptide complexes with class I molecules of the major histocompatibility complex (MHC). However, there is evidence for an exogenous pathway whereby antigens that are not expected to gain access to the cytoplasm are presented on MHC class I molecules. The most dramatic example is the in vivo phenomenon of cross-priming: antigens from donor cells are acquired by bone-marrow-derived host antigen-presenting cells (APCs) and presented on MHC class I molecules. Two unanswered questions concern the identity of this bone-marrow-derived cell and how such antigens are acquired. Here we show that human dendritic cells, but not macrophages, efficiently present antigen derived from apoptotic cells, stimulating class I-restricted CD8+ CTLs. Our findings suggest a mechanism by which potent APCs acquire antigens from tumours, transplants, infected cells, or even self-tissue, for stimulation or tolerization of CTLs.

Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway.

We have explored the role of an activation-induced T cell molecule, 4-1BB (CDw137), in the amplification of tumor immunity by retrovirus-mediated transduction of the 4-1BB ligand (4-1BBL) into tumor cells. Mice inoculated with P815 tumor cells expressing 4-1BBL developed a strong cytotoxic T lymphocyte (CTL) response and long-term immunity against wild-type tumor. The optimal effect of 4-1BBL in CTL stimulation required B7-CD28 interaction since blockade of this interaction by antibodies down-regulated the expression of 4-1BB on T cells and decreased CTL activity. Furthermore, co-expression of 4-1BBL and B7-1 in the poorly immunogenic AG104A sarcoma enhanced the induction of effector CTL and the rejection of the wild-type tumor while neither 4-1BBL nor B7-1 single transfectants were effective, suggesting a synergistic effect between the 4-1BB and the CD28 co-stimulatory pathways. Our results underscore the importance of the 4-1BB T cell stimulation pathway in the amplification of an antitumor immune response.

Antigen is required for the activation of effector activities, whereas interleukin 2 Is required for the maintenance of memory in ovalbumin-specific, CD8+ cytotoxic T lymphocytes.

The mechanisms that maintain memory in T cells are not completely understood. We have investigated the role of antigen and interleukin (IL)-2 in the growth and maintenance of CD8+ T cells using a cytolytic T cell line specific for ovalbumin (OVA)257-264 presented by H-2Kb. This line does not secrete IL-4 or IL-2; hence, stimulation with the OVA-transfected EL4 line (E.G7-OVA) does not induce proliferation without addition of exogenous growth factors. Furthermore, this line can be maintained continuously by weekly addition of irradiated, splenic filler cells and IL-2, with or without E.G7-OVA. Although IL-2 induced proliferation of these cytotoxic T lymphocytes (CTLs), production of interferon gamma and tumor necrosis factor alpha required stimulation of the CTL with E. G7-OVA. The kinetics of lymphokine secretion after stimulation by E. G7-OVA were the same whether the CTL had been maintained with or without antigen (Ag). In addition, both CTL lines killed E.G7-OVA target cells within 4 h. Thus, the effector functions of these CTLs were rapidly induced by T cell receptor (TCR) occupancy. CTLs cultured with or without Ag also served as memory T cells when parked for 100 d in unirradiated, syngeneic recipients without OVA. In the absence of OVA, the precursor frequency was identical in spleens of normal and beta2-microglobulin knockout recipients, but significantly less in IL-2 knockout mice. The decline of memory in the absence of IL-2 supports data from other investigators, suggesting that cell cycling is important to the maintenance of CD8+ T cell memory. These data also suggest that stimulation of OVA-specific CTLs by lymphokines seems to be more important to maintaining memory than stimulation of TCRs by cross-reactive peptides complexed to class I molecules.

Growth stimulation of tumor-specific cytotoxic T lymphocytes on concanavalin a-immobilized carrier beads.

Human tumor-specific CD4(+) cytotoxic T lymphocytes (CTL) were generated against duodenum papilloma cell line TGBC18TKB from HLA type-matched peripheral blood mononuclear cells. Concanavalin A (Con A) immobilized on carrier beads stimulated growth of the CTL in a long-term culture without repeated antigen stimulation, while soluble Con A induced death of the CTL. The CTL exhibited the target-specific cytotoxicity in a more potent manner than those before the long-term culture in the presence of the immobilized Con A. Enhanced expression of the adhesion molecule, CD11b, was observed on the CTL. These results suggest that immobilized Con A will be useful for continuous growth stimulation and large scale expansion of CTL without tumor antigen.

Recognition of gp43 Tumor-Associated Antigen Peptide by both HLA-A2 Restricted CTL Lines and Antibodies from Melanoma Patients

Previously, we detected a 43-kDa tumor-associated antigen (TAA) using the human monoclonal antibody L92, which recognizes the tetramer peptide KYQI. In the present study, cell lines of cytotoxic T lymphocytes (CTL) specific to the gp43 peptide (DLTMKYQIF) were established from peripheral blood lymphocytes (PBL) of melanoma patients. Patients’ PBL (n= 326) of different HLA Class I types were assessed for gp43 CTL activity. CTL specific to gp43 peptide were generated only from HLA-A2 melanoma patients and not normal donors. gp43 CTL recognized gp43 peptide-pulsed autologous BLC and T2 HLA-A2 target cell lines. Furthermore, CTL lines were shown to kill both HLA-A2 autologous and HLA-A2 allogeneic melanoma cell lines, indicating that gp43 peptide can be processed endogenously and presented by melanoma cells as a common TAA. The gp43 CTL lines did not kill normal cells. Specific amino acids of the peptide were shown to be important determinants in stimulation and recognition of CTL. gp43 peptide, recognized by both antibodies and T cells of melanoma patients, is a novel TAA peptide that may play an important role in anti-tumor immunity in human.

Epidermal Dendritic Cells Induce Potent Antigen-Specific CTL-Mediated Immunity

Professional antigen-presenting cells (APCs) are required for the initiation of an immune response. Dendritic cells (DCs) are the most potent APCs identified thus far and can present antigen in the context of co-stimulatory signals required for the stimulation of both primed and naïve T cells. Cytotoxic T lymphocytes (CTLs) are critical to the immune response against tumors or virally infected cells. Optimal stimulation of antigen-specific CTLs is the goal of evolving immunization strategies for the prevention or therapy of viral infections and tumors. Epidermal dendritic cells (eDCs), or Langerhans cells, can present antigens for the stimulation of CD4+ T cell dependent anti-tumor immunity and may play a role in tumor surveillance. The capacity of eDCs to induce tumor-specific CD8+ CTL immunity has not been determined. We have previously shown that DCs derived from bone marrow precursors (BmDCs) under the influence of cytokines can induce protective, antigen-specific CTL-mediated anti-tumor immunity. Here we show that subcutaneous immunization with ovalbumin (OVA) peptide (SIINFEKL(257-264))-pulsed eDCs induced OVA-specific, CD8+ CTLs that lyse the OVA-expressing target. Furthermore, mice vaccinated with OVA peptide-pulsed eDCs were completely protected from subsequent challenge by the OVA-expressing melanoma MO5. The capacity of peptide-pulsed eDCs to induce CTL-mediated immunity is directly dependent on the dose of eDCs administered. Importantly, the APC capacity of eDCs is comparable to that of BmDCs, as mice immunized with eDC populations containing at least as many class II+/B7.2+ cells as populations of BmDCs were equally protected against challenge with MO5. These results demonstrate that eDCs can be potent inducers of antigen-specific CD8+ CTL-mediated immunity. They suggest that eDCs may be important targets for antigen delivery strategies aimed at inducing antiviral or anti-tumor immunity.