Cutaneous T-cell lymphoma (CTCL) is a lymphoproliferative skin disease with limited therapeutic options. Ten CTCL patients were treated with once-weekly intranodal injection of 1 ؋ 10 6 mature monocyte-derived dendritic cells (DCs) pulsed with 100 g/mL tumor lysate protein equivalent and keyhole limpet hemocyanin (50 g/mL). Tumor-specific delayed-type hypersensitivity (

Dendritic cells (DC) are central regulators of immunity. Signal-induced maturation of DCs is assumed to be the starting point for specific immune responses. To further understand this process, we analyzed the alteration of transcript profiles along the time course of CD40 ligand-induced maturation of human myeloid DCs by Affymetrix GeneChip microarrays covering >6800 genes. Besides rediscovery of genes already described as associated with DC maturation proving reliability of the methods used, we identified clusterin as novel maturation marker. Looking across the time course, we observed synchronized kinetics of distinct functional groups of molecules whose temporal coregulation underscores known cellular events during dendritic cell maturation. For example, an early-peaking wave of inflammatory chemokines was followed by a sustained increase of constitutive chemokines and accompanied by slow but continuous induction of survival proteins. After an immediate but transient induction of cytokine-responsive transcripts, there was an increased expression of a group of genes involved in not only the regulation of cytokine effects, but also of transcription in general. Our results demonstrate that microarray studies along time courses combined with real-time PCR not only discover new marker molecules with functional implications, but also dissect the molecular kinetics of biological processes identifying complex pathways of regulation.

Epithelial and endothelial cells expressing the primary Coxsackie virus B adenovirus (Ad) receptor (CAR) and integrin coreceptors are natural targets of human Ad infections. The fiber knob of species A, C, D, E and F Ad serotypes binds CAR by mimicking the CAR-homodimer interface, and the penton base containing arginine-glycine-aspartate (RGD) motifs binds with low affinity to alphav integrins inducing cell activation. Here, we generated seven different genetically modified Ad vectors with RGD sequences inserted into the HI loop of fiber knob. All mutants bound and infected CAR and alphav integrin-positive epithelial cells with equal efficiencies. However, the Ads containing two additional cysteines, both N and C terminals of the RGD sequence (RGD-4C), were uniquely capable of transducing CAR-less hematopoietic and nonhematopoietic human tumor cell lines and primary melanoma cells. Both binding and transduction of RGD-4C Ad were blocked by soluble RGD peptides. Flow cytometry of cell surface integrins and virus binding to CAR-less cells in the presence of function-blocking anti-integrin antibodies indicated that the alphavbeta5 integrin, but not alphavbeta3, alphaIIbbeta3 or beta1,alpha5 or alpha6-containing integrins served as a functional transduction receptor of the RGD-4C Ads. However, in cells with low levels of alphavbeta5 integrin, the function-blocking anti-alphavbeta5 antibodies were not effective, unlike soluble RGD peptides. Collectively, our data demonstrate that the alphavbeta5 integrin is a functional transduction receptor of RGD-4C Ads in the absence of CAR, and that additional RGD receptors are targets of these viruses. The RGD-4C vectors further extend the tropism of Ads towards potential human therapies.

Cancer/testis-antigens (CTA), a novel and expanding family of immunogenic proteins detected by serological screening of recombinant cDNA expression libraries, encompass promising candidate targets for T-cell based immunotherapy. We screened kryo-preserved tissue of cutaneous T cell lymphoma (CTCL, n=36) such as mycosis fungoides (MF, n=17), pleomorphic cutaneous T-cell lymphoma (n=8) and Sezary's syndrome (SS, n= 11) as well as a non-malignant entity (small plaques parapsoriasis, SPP, n=5), for the expression of CTA by RT-PCR and Northern blot hybridization. From a panel of eleven CTA (MAGE-1, MAGE-C1, MAGE-3, BAGE, GAGE, SSX-1, SSX-2, SSX4, SCP-1, NY-ESO-1 and TS85) (HOM-Tes-85), mRNA expression could be detected for SCP-1 in 8/17 MF and 6/8 pleomorphic CTCL patients but was completely absent in small plaques parapsoriasis. SS patients had a more heterogeneous antigen expression pattern: Gage (1/11), MAGE-1 (3/11), MAGE-3 (6/11), MAGE-C1 (5/11), NY-ESO-1 (7/11) and TS85 (5/11), with expression of MAGE-3 confirmed by immunohistochemistry. CTA could provide defined targets for antigen-based vaccination in a high percentage of cases with CTCL. SCP-1 might serve as an additional diagnostic indicator in early and clinically indistinct lesions suspicious for cutaneous T-cell lymphoma.

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