Metabolic profiling of single cells by exploiting NADH and FAD fluorescence via flow cytometry.

The metabolism of different cells within the same microenvironment can differ and dictate physiological or pathological adaptions. Current single-cell analysis methods of metabolism are not label-free. The study introduces a label-free, live-cell analysis method assessing endogenous fluorescence of NAD(P)H and FAD in surface-stained cells by flow cytometry. OxPhos inhibition, mitochondrial uncoupling, glucose exposure, genetic inactivation of glucose uptake and mitochondrial respiration alter the optical redox ratios of FAD and NAD(P)H as measured by flow cytometry. Those alterations correlate strongly with measurements obtained by extracellular flux analysis. Consequently, metabolically distinct live B-cell populations can be resolved, showing that human memory B-cells from peripheral blood exhibit a higher glycolytic flexibility than naïve B cells. Moreover, the comparison of blood-derived B- and T-lymphocytes from healthy donors and rheumatoid arthritis patients unleashes rheumatoid arthritis-associated metabolic traits in human naïve and memory B-lymphocytes. Taken together, these data show that the optical redox ratio can depict metabolic differences in distinct cell populations by flow cytometry.

Selective Expression of the MAPK Phosphatase Dusp9/MKP-4 in Mouse Plasmacytoid Dendritic Cells and Regulation of IFN-ß Production.

Plasmacytoid dendritic cells (pDCs) efficiently produce large amounts of type I IFN in response to TLR7 and TLR9 ligands, whereas conventional DCs (cDCs) predominantly secrete high levels of the cytokines IL-10 and IL-12. The molecular basis underlying this distinct phenotype is not well understood. In this study, we identified the MAPK phosphatase Dusp9/MKP-4 by transcriptome analysis as selectively expressed in pDCs, but not cDCs. We confirmed the constitutive expression of Dusp9 at the protein level in pDCs generated in vitro by culture with Flt3 ligand and ex vivo in sorted splenic pDCs. Dusp9 expression was low in B220(-) bone marrow precursors and was upregulated during pDC differentiation, concomitant with established pDC markers. Higher expression of Dusp9 in pDCs correlated with impaired phosphorylation of the MAPK ERK1/2 upon TLR9 stimulation. Notably, Dusp9 was not expressed at detectable levels in human pDCs, although these displayed similarly impaired activation of ERK1/2 MAPK compared with cDCs. Enforced retroviral expression of Dusp9 in mouse GM-CSF-induced cDCs increased the expression of TLR9-induced IL-12p40 and IFN-ß, but not of IL-10. Conditional deletion of Dusp9 in pDCs was effectively achieved in Dusp9(flox/flox); CD11c-Cre mice at the mRNA and protein levels. However, the lack of Dusp9 in pDC did not restore ERK1/2 activation after TLR9 stimulation and only weakly affected IFN-ß and IL-12p40 production. Taken together, our results suggest that expression of Dusp9 is sufficient to impair ERK1/2 activation and enhance IFN-ß expression. However, despite selective expression in pDCs, Dusp9 is not essential for high-level IFN-ß production by these cells.

A common role for various human truncated adenomatous polyposis coli isoforms in the control of beta-catenin activity and cell proliferation.

The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancer cases, leading to the synthesis of truncated APC products and the stabilization of ß-catenin. Truncated APC is almost always retained in tumour cells, suggesting that it serves an essential function. Here, RNA interference has been used to down-regulate truncated APC in several colorectal cancer cell lines expressing truncated APCs of different lengths, thereby performing an analysis covering most of the mutation cluster region (MCR). The consequences on proliferation in vitro, tumour formation in vivo and the level and transcriptional activity of ß-catenin have been investigated. Down-regulation of truncated APC results in an inhibition of tumour cell population expansion in vitro in 6 cell lines out of 6 and inhibition of tumour outgrowth in vivo as analysed in one of these cell lines, HT29. This provides a general rule explaining the retention of truncated APC in colorectal tumours and defines it as a suitable target for therapeutic intervention. Actually, we also show that it is possible to design a shRNA that targets a specific truncated isoform of APC without altering the expression of wild-type APC. Down-regulation of truncated APC is accompanied by an up-regulation of the transcriptional activity of ß-catenin in 5 out of 6 cell lines. Surprisingly, the increased signalling is associated in most cases (4 out of 5) with an up-regulation of ß-catenin levels, indicating that truncated APC can still modulate wnt signalling through controlling the level of ß-catenin. This control can happen even when truncated APC lacks the ß-catenin inhibiting domain (CiD) involved in targeting ß-catenin for proteasomal degradation. Thus, truncated APC is an essential component of colorectal cancer cells, required for cell proliferation, possibly by adjusting ß-catenin signalling to the "just right" level.

Integration frequency and intermolecular recombination of rAAV vectors in non-human primate skeletal muscle and liver.

The comprehensive characterization of recombinant adeno-associated viral (rAAV) integration frequency and persistence for assessing rAAV vector biosafety in gene therapy is severely limited due to the predominance of episomal rAAV vector genomes maintained in vivo. Introducing rAAV insertional standards (rAIS), we show that linear amplification-mediated (LAM)-PCR and deep sequencing can be used for validated measurement of rAAV integration frequencies. Integration of rAAV2/1 or rAAV2/8, following intramuscular (IM) or regional intravenous (RI) administration of therapeutically relevant vector doses in nine adult non-human primates (NHP), occurs at low frequency between 10(-4) and 10(-5) both in NHP liver and muscle, but with no preference for specific genomic loci. High resolution mapping of inverted terminal repeat (ITR) breakpoints in concatemeric and integrated vector genomes reveals distinct vector recombination hotspots, including large deletions of up to 3 kb. Moreover, retrieval of integrated rAAV genomes indicated approximately threefold increase in liver compared to muscle. This molecular analysis of rAAV persistence in NHP provides a promising basis for a reliable genotoxic risk assessment of rAAV in clinical trials.

Unique requirements for reactivation of virus-specific memory B lymphocytes.

Memory B cells (MBCs) are rapidly activated upon Ag re-exposure in vivo, but the precise requirements for this process are still elusive. To address these requirements, T cell-independent reactivation of MBCs against virus-like particles was analyzed. As few as 25 MBCs are sufficient for a measurable Ab response after adoptive transfer. We found that MBCs were reactivated upon antigenic challenge to normal levels after depletion of macrophages, CD11c(+) dendritic cells, and matured follicular dendritic cells. Furthermore, MBC responses were possible in TNF/lymphotoxin α double-deficient mice after partial normalization of lymphoid architecture by means of long-term reconstitution with wild-type bone marrow. Activation did not occur when chimeric mice, which still lack all lymph nodes and Peyer's patches, were splenectomized prior to MBC transfer. Together with our finding that MBC responses are weak when Ag was administered within minutes after adoptive MBC transfer, these results strongly suggest that MBCs have to occupy specific niches within secondary lymphoid tissue to become fully Ag-responsive. We provide clear evidence that MBCs are not preferentially resident within the splenic marginal zones and show that impaired homing to lymphoid follicles resulted in significantly diminished activation, suggesting that reactivation of MBCs occurred inside lymphoid follicles. Furthermore, comparison of virus-specific MBC T cell-independent reactivation versus primary T cell-independent type II B cell activation revealed unique requirements of MBC activation.

Protection from CMV infection in immunodeficient hosts by adoptive transfer of memory B cells.

Severe disease associated with cytomegalovirus (CMV) infection is still a major problem in patients who undergo transplantation. Support of the patients' immune defense against the virus is a major goal in transplantation medicine. We have used the murine model of CMV (MCMV) to investigate the potential of a cell-based strategy to support the humoral antiviral immune response. Immunocompetent C57BL/6 mice were infected with MCMV, and memory B cells from the immune animals were adoptively transferred into T-cell- and B-cell-deficient RAG-1(-/-) mice. Following MCMV infection, a virus-specific IgG response developed within 4 to 7 days in the recipient animals. Concomitantly, a significant reduction in viral titers and DNA copies in several organs was observed. In addition, the memory B-cell transfer provided long-term protection from the lethal course of the infection that is invariably seen in immunodeficient animals. Transfer of memory B cells was also effective in protecting from an already ongoing viral infection, indicating a therapeutic potential of virus-specific memory B cells. T cells were not involved in this process. Our data provide evidence that a cell-based strategy to support the humoral immune response can be effective to combat infectious pathogens in severely immunodeficient hosts.