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Zusammenfassung

Mimotopes provide an alternative to natural T cell epitopes for cancer immune therapy, as they can recruit and stimulate T cell repertoires that deviate from the repertoires engaged with the tumor and exposed to disease-related immune suppression. Here, mimotopes of a shared tumor-associated T cell epitope in cutaneous lymphoma were tested for their capacities to induce clinical and immunological responses in cancer patients. The mimotope sequences had been determined by a combinatorial peptide library approach without knowledge of the corresponding natural tumor-associated antigen. Vaccination with these mimotopes together with helper T cell-inducing antigens led to complete tumor remission in the two patients tested. After each booster vaccination, enhanced frequencies of mimotope-specific CD8+ T cells were detected in the peripheral blood of the patients, and the CTL proved to be cytotoxic and tumoricidal when tested in vitro. These data provide a first indication of clinical efficacy of mimotopes in cancer patients.

Nef is an accessory protein of human and simian immunodeficiency viruses (HIV and SIV) that is required for efficient viral infectivity and pathogenicity. It decreases the expression of CD4 on the surface of infected cells. V1H is the regulatory subunit H of the vacuolar membrane ATPase (V-ATPase). Previously, the interaction between Nef and V1H has been found to facilitate the internalization of CD4, suggesting that V1H could connect Nef to the endocytic machinery. In this study, we demonstrate that V1H binds to the C-terminal flexible loop in Nef from HIV-1 and to the medium chain (mu2) of the adaptor protein complex 2 (AP-2) in vitro and in vivo. The interaction sites of V1H and mu2 were mapped to a central region in V1H from positions 133 to 363, which contains 4 armadillo repeats, and to the N-terminal adaptin-binding domain in mu2 from positions 1 to 145. Fusing Nef to V1H reproduced the appropriate trafficking of Nef. This chimera internalized CD4 even in the absence of the C-terminal flexible loop in Nef. Finally, blocking the expression of V1H decreased the enhancement of virion infectivity by Nef. Thus, V1H can function as an adaptor for interactions between Nef and AP-2.

The negative factor (Nef) is one of six accessory proteins from primate lentiviruses (HIV-1, HIV-2, and SIV). It leads to high levels of viremia and the progression to AIDS in monkeys and humans. In this study, we demonstrated that Nef from HIV-1 binds to the regulatory subunit (p85) of phosphatidylinositol-3-kinase (PI3K). This interaction depended on the C-terminus of p85 and Nef. Moreover, PI3K was required to activate the Nef-associated p21-activated kinase (PAK). Finally, inhibition of PI3K blocked the activation of PAK and decreased the production of viral particles to levels observed with the Nef-deleted provirus. We conclude that Nef assembles a multiprotein signaling complex which is required for the optimal replication of HIV-1.

FVFTLTVPS was identified as the core sequence of a new major histocompatibility complex class II-restricted T-cell epitope of influenza virus matrix protein. Epitope-specific CD4+T cells were detected in the peripheral blood of patients with frequencies of up to 0.94%, depending on the number of additional terminal amino acids.

Summary Background' Mycosis fungoides is the most frequent T-cell lymphoma of the skin. Despite numerous attempts, no tumour antigens have yet been identified. Only in one case has an idiotype-derived peptide been found to trigger CTL of the respective patient. The identification of natural antigens requires the cultivation of large amounts of tumour cells in vitro, which has been possible in two exceptional cases. The identification of synthetic epitopes for tumour-specific CTL with random peptide libraries can overcome this limitation and is a powerful tool for application in the development of immune therapies for a wide range of patients. Materials and methods: The critical amino acids for the construction of epitopes for the CTCL-specific CTL clone My-La CTL were determined with synthetic peptide libraries in positional scanning OX8 format in a standard 61 chromium release assay. Sixteen different peptides could be synthesized from the combinatoric of these amino acids with the canonical anchor amino acids for MHC binding. These peptides were tested for their capacity to stimulate My-La CTL and PBMC of an HLA-matched CTCL patient. Results: A synthetic epitope could be identified for My-La CTL, which was recognized in a HLA-restricted manner. The response towards this epitope was comparable to the response towards their natural target My-La. Using these synthetic epitopes, T cells of a HLA-matched patient could be induced in vitro and led to the establishment of different cell lines and clones. Some of these lines recognized the peptides as well as the allogenic but HLA-matched tumour cell line My-La, indicating that they are specific for a naturally expressed tumour antigen. Conclusions: The identification of synthetic epitopes for tumour-specific CTL clones can be used for the development of vaccines for immune therapies of cancer; such peptides can be applied inter-individually. Synthetic epitopes must not correspond to the natural ones, but they can be even more potent as stimulation of specific T cells and can be fine-tuned to increase the success of the therapy.

To understand the system of secreted proteins and receptors involved in cell-cell signaling, we produced a comprehensive set of recombinant secreted proteins and the extracellular domains of transmembrane proteins, which constitute most of the protein components of the extracellular space. Each protein was tested in a suite of assays that measured metabolic, growth, or transcriptional responses in diverse cell types. The pattern of responses across assays was analyzed for the degree of functional selectivity of each protein. One of the highly selective proteins was a previously undescribed ligand, designated interleukin-34 (IL-34), which stimulates monocyte viability but does not affect responses in a wide spectrum of other assays. In a separate functional screen, we used a collection of extracellular domains of transmembrane proteins to discover the receptor for IL-34, which was a known cytokine receptor, colony-stimulating factor 1 (also called macrophage colony-stimulating factor) receptor. This systematic approach is thus useful for discovering new ligands and receptors and assessing the functional selectivity of extracellular regulatory proteins.

BackgroundStatins have previously been shown to reduce the in vitro infection of human immunodeficiency virus type 1 (HIV-1) through modulation of Rho GTPase activity and lipid raft formation at the cell surface, as well as by disrupting LFA-1 incorporation into viral particles.Principle findingsHere we demonstrate that treatment of an enriched CD4(+) lymphocyte population with lovastatin (Lov), mevastatin (Mev) and simvastatin (activated and non-activated, Sim(A) and Sim(N), respectively) can reduce the cell surface expression of the CC-chemokine receptor CCR5 (PConclusionsThe results indicate that the modulation of CC-chemokine receptor (CCR5) and CC-chemokine (RANTES) expression levels should be considered as contributing to the anti-viral effects of statins, preferentially inhibiting R5 viruses. This observation, in combination with the immunomodulatory activity exerted by statins, suggests they may possess more potent anti-HIV-1 activity when applied during the early stages of infection or in lowering viral transmission. Alternatively, statin treatment could be considered as a way to modulate immune induction such as during vaccination protocols.

We studied human immunodeficiency virus type 1 (HIV-1) chimeric viruses altering in their gp120 V1V2 and V3 envelope regions to better map which genetic alterations are associated with specific virus phenotypes associated with HIV-1 disease progression. The V1V2 and V3 regions studied were based on viruses isolated from an individual with progressing HIV-1 disease. Higher V3 charges were linked with CXCR4 usage, but only when considered within a specific V1V2 and V3 N-linked glycosylation context. When the virus gained R5X4 dual tropism, irrespective of its V3 charge, it became highly resistant to inhibition by RANTES and highly sensitive to inhibition by SDF-1α. R5 viruses with higher positive V3 charges were more sensitive to inhibition by RANTES, while R5X4 dualtropic viruses with higher positive V3 charges were more resistant to inhibition by SDF-1α. Loss of the V3 N-linked glycosylation event rendered the virus more resistant to inhibition by SDF-1α. The same alterations in the V1V2 and V3 regions influenced the extent to which the viruses were neutralized with soluble CD4, as well as monoclonal antibodies b12 and 2G12, but not monoclonal antibody 2F5. These results further identify a complex set of alterations within the V1V2 and V3 regions of HIV-1 that can be selected in the host via alterations of coreceptor usage, CC/CXC chemokine inhibition, CD4 binding, and antibody neutralization.

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIVmac239for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity.

Mimotopes of a tumor-associated T cell epitope were determined using randomized and combinatorial peptide libraries and a CD8(+) T cell clone specific for the cutaneous T cell lymphoma cell line MyLa. Antigen recognition by this clone was found to be HLA-B8 restricted. More than 80 % of HLA-matched patients with cutaneous T cell lymphoma had mimotope-specific CD8(+) T cells in their peripheral blood. Mimotope-specific T cells isolated and expanded from a patient lysed MyLa cells in in vitro assays thus demonstrating their cytolytic capacity and tumor specificity. Mimotope vaccination of a patient without detectable mimotope-specific T cells induced frequencies of these cells of up to 1.82 % of the peripheral blood CD8(+) T cells.

Cellular signaling downstream of Ras is highly diversified and may involve many different effector molecules. A potential candidate is AF6 which was originally identified as a fusion to ALL-1 in acute myeloid leukemia. In the present work the interaction between Ras and AF6 is characterized and compared with other effectors. The binding characteristics are quite similar to Raf and RalGEF, i.e. nucleotide dissociation as well as GTPase-activating protein activity are inhibited, whereas the intrinsic GTPase activity of Ras is unperturbed by AF6 binding. Particularly, the dynamics of interaction are similar to Raf and RalGEF with a lifetime of the Ras. AF6 complex in the millisecond range. As probed by 31P NMR spectroscopy one of two major conformational states of Ras is stabilized by the interaction with AF6. Looking at the affinities of AF6 to a number of Ras mutants in the effector region, a specificity profile emerges distinct from that of other effector molecules. This finding may be useful in defining the biological function of AF6 by selectively switching off other pathways downstream of Ras using the appropriate effector mutant. Notably, among the Ras-related proteins AF6 binds most tightly to Rap1A which could imply a role of Rap1A in AF6 regulation.