Polyclonal expansion of T cells with the TCR V beta type of the tumour cell in lesions of cutaneous T-cell lymphoma: evidence for possible superantigen involvement.

The involvement of superantigens in the pathology of cutaneous T-cell lymphomas (CTCL) has been suggested before, but without unequivocal evidence for superantigen activity in the patients. Seeking evidence for superantigen activity we analysed clones and microdissected single cells isolated from the epidermis of early-stage lesions of a CTCL patient for their T-cell receptor (TCR) V beta expression and TCR V gamma gene rearrangements. The vast majority of these T cells expressed the TCR V beta family type of the tumour. From their TCR gamma gene rearrangements, however, these cells were polyclonal. The tumour cell clone accounted for about 60% of these cells, about 40% were of heterogeneous origin. This dominance of a single V beta family in the polyclonally expanded dermal T-cell populations implies superantigen activity in the CTCL lesions.

Nef increases infectivity of HIV via lipid rafts.

Lipid rafts, also known as detergent-resistant membranes (DRM), are microdomains in the plasma membrane enriched in sphingolipids and cholesterol (reviewed in [1, 2]). Human immunodeficiency virus 1 (HIV) buds via lipid rafts [3, 4]. However, the targeting of viral structural components to DRM and its consequences for viral replication are not understood. Moreover, the negative factor Nef from HIV increases viral infectivity (reviewed in [5, 6]). With no apparent differences in structural components and morphology between wild-type and DeltaNef virons, the latter viruses display less efficient reverse transcription in target cells. As Nef is expressed abundantly early in the viral replicative cycle [7], we hypothesized that Nef could affect viral morphogenesis and budding to render viruses more infectious. In this report, we demonstrated first that Nef increases viral budding from lipid rafts. Second, in the presence of Nef, viral envelopes contain more ganglioside (GM1), which is a major component of lipid rafts. This finding correlated directly with the increased infectivity of HIV. Finally, the depletion of exogenous and endogenous cholesterol biochemically and genetically, which disrupted lipid rafts, decreased viral infectivity only in the presence of Nef. Importantly, HIV lacking the nef gene remained unaffected by these manipulations. We conclude that lipids in virions are essential for viral infectivity. Thus, HIV becomes more infectious when it buds from lipid rafts, and Nef plays a major role in this process.

Thermodynamics of Ras/effector and Cdc42/effector interactions probed by isothermal titration calorimetry.

Proliferation, differentiation, and morphology of eucaryotic cells is regulated by a large network of signaling molecules. Among the major players are members of the Ras and Rho/Rac subfamilies of small GTPases that bind to different sets of effector proteins. Recognition of multiple effectors is important for communicating signals into different pathways, leading to the question of how an individual GTPase achieves tight binding to diverse targets. To understand the observed specificity, detailed information about binding energetics is expected to complement the information gained from the three-dimensional structures of GTPase/effector protein complexes. Here, the thermodynamics of the interaction of four closely related members of the Ras subfamily with four different effectors and, additionally, the more distantly related Cdc42/WASP couple were quantified by means of isothermal titration calorimetry. The heat capacity changes upon complex formation were rationalized in light of the GTPase/effector complex structures. Changes in enthalpy, entropy, and heat capacity of association with various Ras proteins are similar for the same effector. In contrast, although the structures of the Ras-binding domains are similar, the thermodynamics of the Ras/Raf and Ras/Ral guanine nucleotide dissociation stimulator interactions are quite different. The energy profile of the Cdc42/WASP interaction is similar to Ras/Ral guanine nucleotide dissociation stimulator, despite largely different structures and interface areas of the complexes. Water molecules in the interface cannot fully account for the observed discrepancy but may explain the large range of Ras/effector binding specificity. The differences in the thermodynamic parameters, particularly the entropy changes, could help in the design of effector-specific inhibitors that selectively block a single pathway.

Negative factor from SIV binds to the catalytic subunit of the V-ATPase to internalize CD4 and to increase viral infectivity.

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 SIV(mac239) for 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 for tumor-specific T lymphocytes in human cancer determined with combinatorial peptide libraries.

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.

Detection and quantification of CD4(+) T cells with specificity for a new major histocompatibility complex class II-restricted influenza A virus matrix protein epitope in peripheral blood of influenza patients.

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.

A T-cell epitope determined with random peptide libraries and combinatorial peptide chemistry stimulates T cells specific for cutaneous T-cell lymphoma.

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 61chromium 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.

Structural and biochemical analysis of Ras-effector signaling via RalGDS.

The structure of the complex of Ras with the Ras-binding domain of its effector RalGDS (RGS-RBD), the first genuine Ras-effector complex, has been solved by X-ray crystallography. As with the Rap-RafRBD complex (Nasser et al., 1995), the interaction is via an inter-protein beta-sheet between the switch I region of Ras and the second strand of the RGS-RBD sheet, but the details of the interactions in the interface are remarkably different. Mutational studies were performed to investigate the contribution of selected interface residues to the binding affinity. Gel filtration experiments show that the Ras x RGS-RBD complex is a monomer. The results are compared to a recently determined structure of a similar complex using a Ras mutant (Huang et al., 1998) and are discussed in relation to partial loss-of-function mutations and the specificity of Ras versus Rap binding.

Thermodynamic and kinetic characterization of the interaction between the Ras binding domain of AF6 and members of the Ras subfamily.

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.

Discrimination of amino acids mediating Ras binding from noninteracting residues affecting raf activation by double mutant analysis.

The contribution of residues outside the Ras binding domain of Raf (RafRBD) to Ras-Raf interaction and Ras-dependent Raf activation has remained unresolved. Here, we utilize a double mutant approach to identify complementary interacting amino acids that are involved in Ras-Raf interaction and activation. Biochemical analysis demonstrates that Raf-Arg59 and Raf-Arg67 from RafRBD are interacting residues complementary to Ras-Glu37 located in the Ras effector region. Raf-Arg59 and Raf-Arg67 also mediate interaction with Ras-Glu37 in Ras-dependent Raf activation. The characteristics observed here can be used as criteria for a role of residues from other regions of Raf in Ras-Raf interaction and activation. We developed a quantitative two-hybrid system as a tool to investigate the effect of point mutations on protein-protein interactions that elude biochemical analysis of bacterially expressed proteins. This assay shows that Raf-Ser257 in the RafCR2 domain does not contribute to Ras-Raf interaction and that the Raf-S257L mutation does not restore Raf binding to Ras-E37G. Yet, Raf-S257L displays high constitutive kinase activity and further activation by Ras-G12V/E37G is still impaired as compared with activation by Ras-G12V. This strongly suggests that the RafCR2 domain is an independent domain involved in the control of Raf activity and a common mechanism for constitutively activating mutants may be the interference with the inactive ground state of the kinase.