Erwin S. Schultz

Rapid Induction of Tumor-specific Type 1 T Helper Cells in Metastatic Melanoma Patients by Vaccination with Mature, Cryopreserved, Peptide-loaded Monocyte-derived Dendritic Cells

There is consensus that an optimized cancer vaccine will have to induce not only CD8+ cytotoxic but also CD4+ T helper (Th) cells, particularly interferon (IFN)-γ–producing, type 1 Th cells. The induction of strong, ex vivo detectable type 1 Th cell responses has not been reported to date. We demonstrate now that the subcutaneous injection of cryopreserved, mature, antigen-loaded, monocyte-derived dendritic cells (DCs) rapidly induces unequivocal Th1 responses (ex vivo detectable IFN-γ–producing effectors as well as proliferating precursors) both to the control antigen KLH and to major histocompatibility complex (MHC) class II–restricted tumor peptides (melanoma-antigen [Mage]-3.DP4 and Mage-3.DR13) in the majority of 16 evaluable patients with metastatic melanoma. These Th1 cells recognized not only peptides, but also DCs loaded with Mage-3 protein, and in case of Mage-3DP4–specific Th1 cells IFN-γ was released even after direct recognition of viable, Mage-3–expressing HLA-DP4+ melanoma cells. The capacity of DCs to rapidly induce Th1 cells should be valuable to evaluate whether Th1 cells are instrumental in targeting human cancer and chronic infections.

Tumor‐specific shared antigenic peptides recognized by human T cells

Summary: The first tumor‐specific shared antigens and the cancer‐germline genes that code for these antigens were identified with antitumor cytolytic T lymphocytes obtained from cancer patients. A few HLA class I‐restricted antigenic peptides were identified by this ‘direct approach’. A large set of additional cancer‐germline genes have now been identified by purely genetic approaches or by screening tumor cDNA expression libraries with the serum of cancer patients. As a result, a vast number of sequences are known that can code for tumor‐specific shared antigens, but most of the encoded antigenic peptides have not yet been identified. We review here recent ‘reverse immunology’ approaches for the identification of new antigenic peptides. They are based on in vitro stimulation of naive T cells with dendritic cells that have either been loaded with a cancer‐germline protein or that have been transduced with viruses carrying cancer‐germline coding sequences. These approaches have led to the identification of many new antigenic peptides presented by class I or class II molecules. We also describe some aspects of the processing and presentation of these antigenic peptides.

The Production of a New MAGE-3 Peptide Presented to Cytolytic T Lymphocytes by HLA-B40 Requires the Immunoproteasome

By stimulating human CD8+ T lymphocytes with autologous dendritic cells infected with an adenovirus encoding MAGE-3, we obtained a cytotoxic T lymphocyte (CTL) clone that recognized a new MAGE-3 antigenic peptide, AELVHFLLL, which is presented by HLA-B40. This peptide is also encoded by MAGE-12. The CTL clone recognized MAGE-3–expressing tumor cells only when they were first treated with IFN-γ. Since this treatment is known to induce the exchange of the three catalytic subunits of the proteasome to form the immunoproteasome, this result suggested that the processing of this MAGE-3 peptide required the immunoproteasome. Transfection experiments showed that the substitution of β5i (LMP7) for β5 is necessary and sufficient for producing the peptide, whereas a mutated form of β5i (LMP7) lacking the catalytically active site was ineffective. Mass spectrometric analyses of in vitro digestions of a long precursor peptide with either proteasome type showed that the immunoproteasome produced the antigenic peptide more efficiently, whereas the standard proteasome more efficiently introduced cleavages destroying the antigenic peptide. This is the first example of a tumor-specific antigen exclusively presented by tumor cells expressing the immunoproteasome.

Generation of an Optimized Polyvalent Monocyte-Derived Dendritic Cell Vaccine by Transfecting Defined RNAs after Rather Than before Maturation

Transfection with RNA is an attractive method of Ag delivery to dendritic cells (DCs), but has not yet been standardized. We describe in this study the methods to efficiently generate an optimized mature monocyte-derived DC vaccine at clinical scale based on the electroporation of several RNAs either into immature DC followed by maturation or, alternatively, directly into mature DCs, which has not been possible so far with such high efficiency. Electroporation of DCs resulted in high yield, high transfection efficiency (>90%), and high migration capacity. Intracellular staining allowed the study of the expression kinetics of Ags encoded by the transfected RNAs (MelanA, MAGE-3, and survivin) and a validation of the vaccine (≥90% transfection efficiency). Expression of all three Ags peaked 3–4 h after electroporation in DC transfected either before or after maturation, but decreased differently. The DC vaccine can also be cryopreserved and nevertheless retains its viability, stimulatory capacity as well as migratory activity. In addition, we uncover that DC transfected after rather than before maturation appear to be preferable vaccines not only from a production point of view but also because they appear to be immunologically superior for CTL induction in sharp contrast to common belief. DCs transfected after maturation not only more effectively generate and present the Mage-3.A1 and MelanA.A2.1 epitopes to T cell clones, but they even are superior in priming to the standard proteasome-dependent MelanA.A2.1 wild-type prototype tumor epitope, both in terms of T cell expansion and effector function on a per cell basis.

T cell recognition of desmoglein 3 peptides in patients with pemphigus vulgaris and healthy individuals

Pemphigus vulgaris is a severe autoimmune disease caused by autoantibodies against the cutaneous adhesion molecule, desmoglein 3 (Dsg3). The aim of this study was to characterize the specificity of autoreactive Th cells, which presumably regulate Dsg3-specific autoantibody production. Ninety-seven Th1 and Th2 clones isolated from 16 pemphigus patients and 12 HLA-matched healthy donors recognized the Dsg3 peptides, DG3(78-94), DG3(96-112), DG3(189-205), DG3(205-221), and DG3(250-266). Peptide DG3(96-112), and to a lesser extent DG3(250-266), was recognized by the majority of T cells from patients and healthy donors in association with HLA-DRB1*0402 and DQB1*0503 which were prevalent in the pemphigus patients and Dsg3-responsive healthy donors. Analyzing the Vβ-chain of the TCR of the DG3(96-112)-specific T cells showed no restricted TCR usage. Peptides DG3(342-358) and DG3(376-392) were exclusively recognized by T cell clones (n = 13) from patients while DG3(483-499) was only recognized by T cell clones (n = 3) from a healthy donor. All Dsg3 peptides contained conserved amino acids at relative positions 1, 4, and 6; amino acids with a positive charge at position 4 presumably represent anchor motifs for DRB1*0402. These findings demonstrate that T cell recognition of distinct Dsg3 peptides is restricted by distinct HLA class II molecules and is independent from the development of pemphigus vulgaris.

Antigen loading of dendritic cells with whole tumor cell preparations

Dendritic cells (DC) based vaccinations have been widely used for the induction of anti-tumoral immunity in clinical studies. Antigen loading of DC with whole tumor cell preparations is an attractive method whenever tumor cell material is available. In order to determine parameters for the loading procedure, we performed dose finding and timing experiments. We found that apoptotic and necrotic melanoma cells up to a ratio of one-to-one, equivalent to 1mg/ml protein per 1 x 10(6) DC, can be added to monocyte derived DC without effecting DC recovery extensively. Using the isolated protein content of tumor cells (lysate) as a parameter, up to 5 mg/ml protein per 1 x 10(6) DC can be added. To achieve significant protein uptake at least 1 mg/ml of protein have to be added for more than 24 h as tested with FITC-labelled ovalbumin. Maturation inducing cytokines can be added simultaneously with the tumor cell preparations to immature DC without affecting the uptake. Furthermore, we tested the feasibility of cryopreservation of loaded and matured DC to facilitate the generation of ready to use aliquots. DC were cryopreserved in a mix of human serum albumin, DMSO and 5% glucose. After thawing, surface expression of molecules indicating the mature status (CD83, costimulatory and MHC molecules), was found to be unaltered. Furthermore, cryopreserved DC kept the capability to stimulate allogenic T-cell proliferation in mixed leukocyte reactions at full level. Loaded and matured DC pulsed with influenza matrix peptide (IMP) retained the capacity to induce the generation of IMP-specific cytotoxic T-lymphocytes after cryopreservation as measured by ELISPOT and tetramer staining. The expression of the chemokine receptor CXCR-4 and CCR-7 remained unaltered during cryopreservation and the migratory responsiveness towards MIP-3beta was unaltered as measured in a migration assay. Thus we conclude that the large scale loading and maturation of DC with whole tumor cell preparations can be performed in a single session. These data will facilitate the clinical application of DC loaded with whole tumor cell preparations.

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-3243–258), 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.

Dendritic Cell-Based Immunotherapy

Dendritic cell (DC)-based vaccinations represent a promising approach for the immunotherapy of cancer and infectious diseases as DCs play an essential role in initiating cellular immune responses. A number of clinical trials using ex vivo-generated DCs have been performed so far and only minor toxicity has been reported. Both the induction of antigen-specific T cells and clinical responses have been observed in vaccinated cancer patients. Nevertheless, DC-based immunotherapy is still in its infancy and there are many issues to be addressed such as antigen loading procedures, DC source and maturational state, migration properties, route, frequency, and dosage of DC vaccination. The increasing knowledge of DC biology should be used to improve the efficacy of this new therapy.

Pimecrolimus 1% Cream in the Treatment of Facial Psoriasis : A 16-Week Open-Label Study

Background: Facial psoriasis requires a treatment approach other than topical corticosteroids which bear the risk of skin atrophy. Topical pimecrolimus has been shown to be effective in atopic eczema and recently in psoriasis. Objective: The aim of this open-label single-center investigator-initiated study was to evaluate the efficacy and safety of pimecrolimus 1% cream in patients with facial psoriasis. Methods: 20 adults with facial psoriasis were enrolled. Pimecrolimus 1% cream was applied twice daily to psoriatic lesions of the face over an 8-week period. An 8-week follow-up was added. Results: All clinical parameters showed a significant improvement after 8 and 16 weeks compared to baseline. Pimecrolimus 1% cream was effective and well tolerated. Conclusions: This is the first clinical study with a larger patient cohort reporting a relevant therapeutic effect and favorable safety profile of pimecrolimus 1% cream in facial psoriasis.

A MAGE-1 peptide recognized on HLA-DR15 by CD4(+) T cells.

Antigens encoded by MAGE genes and recognized by T cells are of interest for cancer immunotherapy because of their strict tumoral specificity and because they are shared by many tumors. Several MAGE‐1 peptide that are recognized by CD8+ cytolytic T lymphocytes have been used in therapeutic vaccination trials. To obtain anti‐tumor immune response, vaccines combining peptides recognized by CD8+ and peptides recognized by CD4+ T cells might be optimal. We focused therefore on the identification of MAGE peptides recognized by CD4+ T cells. We report here the identification of MAGE‐1 epitope EYVIKVSARVRF, which is presented to CD4+ T lymphocytes by HLA‐DR15. This HLA allele is present in 29 % of Asians and 17 % of Caucasians.