IL-15 augments antitumoral activity of an ErbB2/HER2 cancer vaccine targeted to professional antigen-presenting cells

Targeted delivery of tumor-associated antigens to professional antigen-presenting cells (APC) is being explored as a strategy to enhance the antitumoral activity of cancer vaccines. Here, we generated a cell-based system for continuous in vivo production of a CTLA-4-ErbB2 fusion protein as a therapeutic vaccine. The chimeric CTLA-4-ErbB2 molecule contains the extracellular domain of CTLA-4 for specific targeting to costimulatory B7 molecules on the surface of APC, genetically fused to residues 1-222 of human ErbB2 (HER2) as an antigenic determinant. In wild-type BALB/c mice, inoculation of syngeneic epithelial cells continuously secreting the CTLA-4-ErbB2 fusion vaccine in the vicinity of subcutaneously growing ErbB2-expressing renal cell carcinomas resulted in the rejection of established tumors, accompanied by the induction of ErbB2-specific antibodies and cytotoxic T cells. In contrast, treatment with CTLA-4-ErbB2 vaccine-secreting producer cells alone was insufficient to induce tumor rejection in ErbB2-transgenic WAP-Her-2 F1 mice, which are characterized by pronounced immunological tolerance to the human self-antigen. When CTLA-4-ErbB2 producer cells were modified to additionally secrete interleukin (IL)-15, antigen-specific antitumoral activity of the vaccine in WAP-Her-2 F1 mice was restored, documented by an increase in survival, and marked inhibition of the growth of established ErbB2-expressing, but not antigen-negative tumors. Our results demonstrate that continuous in vivo expression of an APC-targeted ErbB2 fusion protein results in antigen-specific immune responses and antitumoral activity in tumor-bearing hosts, which is augmented by the pleiotropic cytokine IL-15. This provides a rationale for further development of this approach for specific cancer immunotherapy.     

Some peculiar synaptic complexes in the first visual ganglion of the fly,Musca domestica

Summary In the lamina ganglionaris, the first optic ganglion of the fly, the inventory of cell types as well as the patterns of their connections are well known from light microscopic investigations. Even the synaptic contacts are known with relative completeness. However, the structural details visible on electron micrographs are very difficult to interpret in functional terms. This paper concentrates on two aspects: 1) the synaptic complex between a retinula cell axon and four postsynaptic elements, arranged in a constant elongated array (it is suggested that all synapses in which the retinula cell is presynaptic are of this kind), and 2) the gnarl complex in which a presynaptic specialization in one neuron is separated from another neuron by a complicated glial invagination. The participation of glia at postsynaptic sites seems to be quite common in this ganglion. Occasionally it seems that a glia cell is the only postsynaptic partner facing a presynaptic specialization within a neuron.     

Superantigen-induced T cell:B cell conjugation is mediated by LFA-1 and requires signaling through Lck, but not ZAP-70.

The formation of a conjugate between a T cell and an APC requires the activation of integrins on the T cell surface and remodeling of cytoskeletal elements at the cell-cell contact site via inside-out signaling. The early events in this signaling pathway are not well understood, and may differ from the events involved in adhesion to immobilized ligands. We find that conjugate formation between Jurkat T cells and EBV-B cells presenting superantigen is mediated by LFA-1 and absolutely requires Lck. Mutations in the Lck kinase, Src homology 2 or 3 domains, or the myristoylation site all inhibit conjugation to background levels, and adhesion cannot be restored by the expression of Fyn. However, ZAP-70-deficient cells conjugate normally, indicating that Lck is required for LFA-1-dependent adhesion via other downstream pathways. Several drugs that inhibit T cell adhesion to ICAM-1 immobilized on plastic, including inhibitors of mitogen-activated protein/extracellular signal-related kinase kinase, phosphatidylinositol-3 kinase, and calpain, do not inhibit conjugation. Inhibitors of phospholipase C and protein kinase C block conjugation of both wild-type and ZAP-70-deficient cells, suggesting that a phospholipase C that does not depend on ZAP-70 for its activation is involved. These results are not restricted to Jurkat T cells; Ag-specific primary T cell blasts behave similarly. Although the way in which Lck signals to enhance LFA-1-dependent adhesion is not clear, we find that cells lacking functional Lck fail to recruit F-actin and LFA-1 to the T cell:APC contact site, whereas ZAP-70-deficient cells show a milder phenotype characterized by disorganized actin and LFA-1 at the contact site.     

454 Genome sequencing of Pseudoperonospora cubensis reveals effector proteins with a QXLR translocation motif

Pseudoperonospora cubensis is a biotrophic oomycete pathogen that causes downy mildew of cucurbits, a devastating foliar disease threatening cucurbit production worldwide. We sequenced P. cubensis genomic DNA using 454 pyrosequencing and obtained random genomic sequences covering approximately 14% of the genome, thus providing the first set of useful genomic sequence information for P. cubensis. Using bioinformatics approaches, we identified 32 putative RXLR effector proteins. Interestingly, we also identified 29 secreted peptides with high similarity to RXLR effectors at the N-terminal translocation domain, yet containing an R-to-Q substitution in the first residue of the translocation motif. Among these, a family of QXLR-containing proteins, designated as PcQNE, was confirmed to have a functional signal peptide and was further characterized as being localized in the plant nucleus. Internalization of secreted PcQNE into plant cells requires the QXLR-EER motif. This family has a large number of near-identical copies within the P. cubensis genome, is under diversifying selection at the C-terminal domain, and is upregulated during infection of plants, all of which are common characteristics of characterized oomycete effectors. Taken together, the data suggest that PcQNE are bona fide effector proteins with a QXLR translocation motif, and QXLR effectors are prevalent in P. cubensis. Furthermore, the massive duplication of PcQNE suggests that they might play pivotal roles in pathogen fitness and pathogenicity.     

The actin regulatory protein HS1 is required for antigen uptake and presentation by dendritic cells

The hematopoietic actin regulatory protein hematopoietic lineage cell-specific protein 1 (HS1) is required for cell spreading and signaling in lymphocytes, but the scope of HS1 function in Ag presentation has not been addressed. We show that dendritic cells (DCs) from HS1(-/-) mice differentiate normally and display normal LPS-induced upregulation of surface markers and cytokines. Consistent with their normal expression of MHC and costimulatory molecules, HS1(-/-) DCs present OVA peptide efficiently to CD4(+) T cells. However, presentation of OVA protein is defective. Similarly, MHC class I-dependent presentation of VSV8 peptide to CD8(+) T cells occurs normally, but cross-presentation of GRP94/VSV8 complexes is defective. Analysis of Ag uptake pathways shows that HS1 is required for receptor-mediated endocytosis, but not for phagocytosis or macropinocytosis. HS1 interacts with dynamin 2, a protein involved in scission of endocytic vesicles. However, HS1(-/-) DCs showed decreased numbers of endocytic invaginations, whereas dynamin-inhibited cells showed accumulation of these endocytic intermediates. Taken together, these studies show that HS1 promotes an early step in the endocytic pathway that is required for efficient Ag presentation of exogenous Ag by DCs.     

Structural and functional properties of proteasome activator PA28

The proteasome activator PA28 or 11S regulator is a protein complex composed of two different but homologous polypeptides, termed PA28 and PA28. The purified activator protein (_200 kDa) is a ring-shaped heteromultimer containing the two polypeptides, possibly with an _{3 3} stoichiometry. The activator, which by itself shows no hydrolytic activity elicits activation of the proteasome's multiple peptidase activities by binding to the terminal rings of the proteinase. In vitro, active PA28 can be reconstituted from isolated and subunits, yielding two different oligomers: with the single subunit, PA28 homomultimers with moderate stimulatory activity toward 20S proteasomes are obtained whereas isolated -subunits are unable to form oligomers and are devoid of stimulatory activity. However, in the presence of both subunits, heteromultimers form, concomitant with restoration of full stimulatory activity. The recent finding that PA28 modulates the proteasome-catalyzed production of antigenic peptides presented to the immune system on MHC class I molecules indicates a cellular function of the activator in antigen processing. Abbreviations: IFN – interferon; LMP – low molecular weight peptide; MHC – major histocompatibility complex.     

Complex determinants in human immunodeficiency virus type 1 envelope gp120 mediate CXCR4-dependent infection of macrophages

Host cell range, or tropism, combined with coreceptor usage defines viral phenotypes as macrophage tropic using CCR5 (M-R5), T-cell-line tropic using CXCR4 (T-X4), or dually lymphocyte and macrophage tropic using CXCR4 alone or in combination with CCR5 (D-X4 or D-R5X4). Although envelope gp120 V3 is necessary and sufficient for M-R5 and T-X4 phenotypes, the clarity of V3 as a dominant phenotypic determinant diminishes in the case of dualtropic viruses. We evaluated D-X4 phenotype, pathogenesis, and emergence of D-X4 viruses in vivo and mapped genetic determinants in gp120 that mediate use of CXCR4 on macrophages ex vivo. Viral quasispecies with D-X4 phenotypes were associated significantly with advanced CD4+-T-cell attrition and commingled with M-R5 or T-X4 viruses in postmortem thymic tissue and peripheral blood. A D-X4 phenotype required complex discontinuous genetic determinants in gp120, including charged and uncharged amino acids in V3, the V5 hypervariable domain, and novel V1/V2 regions distinct from prototypic M-R5 or T-X4 viruses. The D-X4 phenotype was associated with efficient use of CXCR4 and CD4 for fusion and entry but unrelated to levels of virion-associated gp120, indicating that gp120 conformation contributes to cell-specific tropism. The D-X4 phenotype describes a complex and heterogeneous class of envelopes that accumulate multiple amino acid changes along an evolutionary continuum. Unique gp120 determinants required for the use of CXCR4 on macrophages, in contrast to cells of lymphocytic lineage, can provide targets for development of novel strategies to block emergence of X4 quasispecies of human immunodeficiency virus type 1.     

CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells

CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.     

Requirement of Indoleamines and a V-type Proton ATPase for the Expression of the Circadian Glow Rhythm in Gonyaulax polyedra

In the dinoflagellate Gonyaulax polyedra, bioluminescence is known to be controlled by proton transfer from an acidic vacuole system to the scintillons. We demonstrate that bafilomycin A 1, a specific blocker of V-type proton ATPases, inhibits at low concentrations (down to 2 × 10 –8 M) bioluminescence and, in particular, the circadian glow maximum. For many hours bafilomycin A 1 does not interfere with the capacity of the bioluminescent system. Therefore, we conclude on the participation of a V-type ATPase in proton accumulation in the acidic vacuoles. Inhibition of tryptophan hydroxylase by p-chlorophenylalanine, p-fluorophenylalanine, or 5-fluorotryptophan also suppresses the circadian glow maximum. After inhibition of the enzyme by p-chlorophenylalanine, the glow peak can be restored, without any additional unspecific effects on bioluminescence, by supplementation with 5-hydroxytryptophan. Therefore, the availability of indoleamines is required for the expression of the glow maximum. Since 5-methoxytryptamine is the only physiologically occurring indoleamine with substantial effects on bioluminescence at low concentrations (below 10 –7 M), and since this substance accumulates in the second half of the night to stimulatory concentrations, this indolic metabolite may represent the physiologically active substance involved in the expression of the glow maximum.