Accounting Problems Under the Excess Profits Tax

DNA vaccines based on subunits from pathogens have several advantages over other vaccine strategies. DNA vaccines can easily be modified, they show good safety profiles, are stable and inexpensive to produce, and the immune response can be focused to the antigen of interest. However, the immunogenicity of DNA vaccines which is generally quite low needs to be improved. Electroporation and co-delivery of genetically encoded immune adjuvants are two strategies aiming at increasing the efficacy of DNA vaccines. Here, we have examined whether targeting to antigen-presenting cells (APC) could increase the immune response to surface envelope glycoprotein (Env) gp120 from Human Immunodeficiency Virus type 1 (HIV- 1). To target APC, we utilized a homodimeric vaccine format denoted vaccibody, which enables covalent fusion of gp120 to molecules that can target APC. Two molecules were tested for their efficiency as targeting units: the antibody-derived single chain Fragment variable (scFv) specific for the major histocompatilibility complex (MHC) class II I-E molecules, and the CC chemokine ligand 3 (CCL3). The vaccines were delivered as DNA into muscle of mice with or without electroporation. Targeting of gp120 to MHC class II molecules induced antibodies that neutralized HIV-1 and that persisted for more than a year after one single immunization with electroporation. Targeting by CCL3 significantly increased the number of HIV-1 gp120-reactive CD8(+) T cells compared to non-targeted vaccines and gp120 delivered alone in the absence of electroporation. The data suggest that chemokines are promising molecular adjuvants because small amounts can attract immune cells and promote immune responses without advanced equipment such as electroporation.Funding Agencies|Research Council of Norway; Odd Fellow</p

MICAL-L1-related and unrelated mechanisms underlying elongated tubular endosomal network (ETEN) in human dendritic cells

The endosomal pathway constitutes a highly dynamic intracellular transport system, which is composed of vesicular and tubular compartments. Endosomal tubules enable geometry-based discrimination between membrane and luminal content. Extended tubular endosomes were suggested to deliver a steady stream of membrane proteins to one location more reliable and effective than vesicular endosomes. Recently, we demonstrated that human dendritic cells (DCs) form a large elongated tubular endosomal network, e.g. ETEN, upon distinct triggers. LPS-stimulation triggered late endosomal tubulation. Additional clustering of class I MHC and ICAM-1 by a cognate interaction between antigen-laden DC and antigen-specific CD8+ T-cells induces formation of transferrin-positive tubules emanating from the endosomal recycling compartment (ERC). We here discuss cell-biological mechanisms that are involved in membrane bending and possibly underlie initiation, elongation, and stabilization of ETEN in human DCs. Using a knock-down approach we demonstrate that MICAL-L1 is necessary for ETEN remodeling originating from ERC in human DCs. </p

Tubulation of endosomal structures in human dendritic cells by Toll-like receptor ligation and lymphocyte contact accompanies antigen cross-presentation

Mouse dendritic cells (DCs) can rapidly extend their Class II MHC-positive late endosomal compartments into tubular structures, induced by Toll-like receptor (TLR) triggering. Within antigen-presenting DCs, tubular endosomes polarize toward antigen-specific CD4+ T cells, which are considered beneficial for their activation. Here we describe that also in human DCs, TLR triggering induces tubular late endosomes, labeled by fluorescent LDL. TLR triggering was insufficient for induced tubulation of transferrin-positive endosomal recycling compartments (ERCs) in human monocyte-derived DCs. We studied endosomal remodeling in human DCs in co-cultures of DCs with CD8+ T cells. Tubulation of ERCs within human DCs requires antigen-specific CD8+ T cell interaction. Tubular remodeling of endosomes occurs within 30 min of T cell contact and involves ligation of HLA-A2 and ICAM-1 by T cell-expressed T cell receptor and LFA-1, respectively. Disintegration of microtubules or inhibition of endosomal recycling abolished tubular ERCs, which coincided with reduced antigen-dependent CD8+ T cell activation. Based on these data, we propose that remodeling of transferrin-positive ERCs in human DCs involves both innate and T cell-derived signals

Dysfunctional BLK in common variable immunodeficiency perturbs B-cell proliferation and ability to elicit antigen-specific CD4+ T-cell help

Common Variable Immunodeficiency (CVID) is the most prevalent primary antibody deficiency, and characterized by defective generation of high-affinity antibodies. Patients have therefore increased risk to recurrent infections of the respiratory and intestinal tract. Development of high-affinity antigen-specific antibodies involves two key actions of B-cell receptors (BCR): transmembrane signaling through BCR-complexes to induce B-cell differentiation and proliferation, and BCR-mediated antigen internalization for class-II MHC-mediated presentation to acquire antigen-specific CD4+ T-cell help. We identified a variant (L3P) in the B-lymphoid tyrosine kinase (BLK) gene of 2 related CVID-patients, which was absent in healthy relatives. BLK belongs to the Src-kinases family and involved in BCR-signaling. Here, we sought to clarify BLK function in healthy human B-cells and its association to CVID. BLK expression was comparable in patient and healthy B-cells. Functional analysis of L3P-BLK showed reduced BCR crosslinking-induced Syk phosphorylation and proliferation, in both primary B-cells and B-LCLs. B-cells expressing L3P-BLK showed accelerated destruction of BCR-internalized antigen and reduced ability to elicit CD40L-expression on antigen-specific CD4+ T-cells. In conclusion, we found a novel BLK gene variant in CVID-patients that causes suppressed B-cell proliferation and reduced ability of B-cells to elicit antigen-specific CD4+ T-cell responses. Both these mechanisms may contribute to hypogammaglobulinemia in CVID-patients

Fcγ receptor antigen targeting potentiates cross-presentation by human blood and lymphoid tissue BDCA-3+ dendritic cells

The reactivation of human cytomegalovirus (HCMV) poses a serious health threat to immune compromised individuals. As a treatment strategy, dendritic cell (DC) vaccination trials are ongoing. Recent work suggests that BDCA-3+ (CD141+) subset DCs may be particularly effective in DC vaccination trials. BDCA-3+ DCs had however been mostly characterized for their ability to cross-present antigen from necrotic cells. We here describe our study of human BDCA-3+ DCs in elicitation of HCMV-specific CD8+ T-cell clones. We show that Fcgamma-receptor (FcγR) antigen targeting facilitates antigen cross-presentation in several DC subsets, including BDCA-3+ DCs. FcγR antigen targeting stimulates antigen uptake by BDCA-1+ rather than BDCA-3+ DCs. Conversely, BDCA-3+ DCs and not BDCA-1+ DCs show improved cross-presentation by FcγR targeting, as measured by induced release of IFNγ and TNF by antigen-specific CD8+ T cells. FcγR-facilitated cross-presentation requires antigen processing in both an acidic endosomal compartment and by the proteasome, and did not induce substantial DC maturation. FcγRII is the most abundantly expressed FcγR on both BDCA-1+ and BDCA-3+ DCs. Furthermore we show that BDCA-3+ DCs express relatively more stimulatory FcγRIIa than inhibitory FcγRIIb in comparison with BDCA-1+ DCs. These studies support the exploration of FcγR antigen targeting to BDCA-3+ DCs for human vaccination purposes

Single-cell glycolytic activity regulates membrane tension and HIV-1 fusion

There has been resurgence in determining the role of host metabolism in viral infection yet deciphering how the metabolic state of single cells affects viral entry and fusion remains unknown. Here, we have developed a novel assay multiplexing genetically-encoded biosensors with single virus tracking (SVT) to evaluate the influence of global metabolic processes on the success rate of virus entry in single cells. We found that cells with a lower ATP:ADP ratio prior to virus addition were less permissive to virus fusion and infection. These results indicated a relationship between host metabolic state and the likelihood for virus-cell fusion to occur. SVT revealed that HIV-1 virions were arrested at hemifusion in glycolytically-inactive cells. Interestingly, cells acutely treated with glycolysis inhibitor 2-deoxyglucose (2-DG) become resistant to virus infection and also display less surface membrane cholesterol. Addition of cholesterol in these in glycolytically-inactive cells rescued the virus entry block at hemifusion and enabled completion of HIV-1 fusion. Further investigation with FRET-based membrane tension and membrane order reporters revealed a link between host cell glycolytic activity and host membrane order and tension. Indeed, cells treated with 2-DG possessed lower plasma membrane lipid order and higher tension values, respectively. Our novel imaging approach that combines lifetime imaging (FLIM) and SVT revealed not only changes in plasma membrane tension at the point of viral fusion, but also that HIV is less likely to enter cells at areas of higher membrane tension. We therefore have identified a connection between host cell glycolytic activity and membrane tension that influences HIV-1 fusion in real-time at the single-virus fusion level in live cells

Fcγ receptor antigen targeting potentiates cross-presentation by human blood and lymphoid tissue BDCA-3+ dendritic cells

The reactivation of human cytomegalovirus (HCMV) poses a serious health threat to immune compromised individuals. As a treatment strategy, dendritic cell (DC) vaccination trials are ongoing. Recent work suggests that BDCA-3+ (CD141+) subset DCs may be particularly effective in DC vaccination trials. BDCA-3+ DCs had however been mostly characterized for their ability to cross-present antigen from necrotic cells. We here describe our study of human BDCA-3+ DCs in elicitation of HCMV-specific CD8+ T-cell clones. We show that Fcgamma-receptor (FcγR) antigen targeting facilitates antigen cross-presentation in several DC subsets, including BDCA-3+ DCs. FcγR antigen targeting stimulates antigen uptake by BDCA-1+ rather than BDCA-3+ DCs. Conversely, BDCA-3+ DCs and not BDCA-1+ DCs show improved cross-presentation by FcγR targeting, as measured by induced release of IFNγ and TNF by antigen-specific CD8+ T cells. FcγR-facilitated cross-presentation requires antigen processing in both an acidic endosomal compartment and by the proteasome, and did not induce substantial DC maturation. FcγRII is the most abundantly expressed FcγR on both BDCA-1+ and BDCA-3+ DCs. Furthermore we show that BDCA-3+ DCs express relatively more stimulatory FcγRIIa than inhibitory FcγRIIb in comparison with BDCA-1+ DCs. These studies support the exploration of FcγR antigen targeting to BDCA-3+ DCs for human vaccination purposes

The Bardet–Biedl syndrome complex component BBS1 controls T cell polarity during immune synapse assembly

Components of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are co-opted by the non-ciliated T cell to orchestrate polarized endosome recycling and to sustain signaling during immune synapse formation. Here, we investigated the potential role of Bardet–Biedl syndrome 1 protein (BBS1), an essential core component of the BBS complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We demonstrated that BBS1 allows for centrosome polarization towards the immune synapse. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH1 (also known as WASHC1), a process that we demonstrated to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that functions upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling

Distinct mechanisms regulate Lck spatial organization in activated T cells

Phosphorylation of the T cell receptor (TCR) by the kinase Lck is the first detectable signaling event upon antigen engagement. The distribution of Lck within the plasma membrane, its conformational state, kinase activity, and protein–protein interactions all contribute to determine how efficiently Lck phosphorylates the engaged TCR. Here, we used cross-correlation raster image correlation spectroscopy and photoactivated localization microscopy to identify two mechanisms of Lck clustering: an intrinsic mechanism of Lck clustering induced by locking Lck in its open conformation and an extrinsic mechanism of clustering controlled by the phosphorylation of tyrosine 192, which regulates the affinity of Lck SH2 domain. Both mechanisms of clustering were differently affected by the absence of the kinase Zap70 or the adaptor Lat. We further observed that the adaptor TSAd bound to and promoted the diffusion of Lck when it is phosphorylated on tyrosine 192. Our data suggest that while Lck open conformation drives aggregation and clustering, the spatial organization of Lck is further controlled by signaling events downstream of TCR phosphorylation