MOESM4 of Validation of an IFNÎł/IL2 FluoroSpot assay for clinical trial monitoring

Additional file 4: Table S4. Diagnostic specificity and sensitivity of the EBV-specific IFNÎł/IL2 FluoroSpot

Effect of N-linked glycosylation on NTCP plasma membrane expression.

<p>Plasma membrane expression was assessed by surface-biotinylation of parental HepG2 cells or those expressing NTCP-WT, NTCP-N5Q, NTCP-N11Q, NTCP-N5,11Q. (A) Representative immunoblot for NTCP membrane expression and for the Transferrin receptor (TfR), as a loading control. Molecular mass is given in kDa on the left-hand side. (B) NTCP expression at the plasma membrane was semi-quantified by Myrcludex-B-FITC intensity. Fluorescence of the parental cells was subtracted before normalization and the net fluorescence units expressed relative to NTCP-WT. *Indicates significant different from NTCP-WT (p<0,05) (C) Confocal microscopic images of HepG2 cells expressing NTCP-WT or mutant proteins stained with anti-HA (green) and counterstained with Hoechst (blue). Scale bar represents 10μm.</p

Treatment with Bafilomycin but not with MG132 induces expression of NTCP-N5,11Q.

<p>(A,B) Parental HepG2 or cells expressing NTCP-WT or NTCP-N5,11Q were treated with 0.1% DMSO, Bafilomycin (10nM/24h), MG132 (20μM/6h) or a combination of Bafilomycin (10nM/24h) and MG132 (20μM/ for the last 6h). (A) Immunofluorescence microscopy of NTCP-WT and NTCP-N5,11Q. in which NTCP was visualized using anti-HA antibody (green) and Hoechst (blue) was used to visualize the nucleus. (B) Results of TCA uptake in parental HepG2 cells (grey) and HepG2 cells expressing NTCP-N5,11Q (black). (C) Results of TCA uptake in HepG2 cells expressing NTCP-WT (grey) or NTCP-N5,11Q (black). (B,C)The uptake capacity of the NTCP was determined as pmol TCA uptake/well. Bars represent the mean +/- sd of three independent experiments, each performed in quadruplicate. *Indicates significant different from DMSO treated (p <0.05). # indicates significant different between the cell-lines in the same treatment (p<0.05).</p

HBV infection of wild type and glycosylation deficient NTCP variants.

<p>HepG2 cells stably expressing NTCP-WT, NTCP-N5Q, NTCP-N11Q or NTCP-N5,11Q were infected with HBV. (A) 3 days post infection, HBeAg production was measured by ELISA. (B) Establishment of HBV infection was determined by quantification of intracellular cccDNA 3 days post-infection. Values are given as mean +/- sd of two independent experiments each performed in triplicate. The dotted line represents the values obtained after infecting parental HepG2 cells that lack NTCP expression. *Significantly different from NTCP-WT values (p < 0.05).</p

Effect of N-linked glycosylation on NTCP protein expression.

<p>(A and B) HA-tagged NTCP was immunoprecipitated (IP) from lysates of HepG2 cells stably expressing NTCP-WT, NTCP-N5Q, NTCP-N11Q or NTCP-N5,11Q. IP samples were subjected to immunoblotting for NTCP (using anti-HA-hrp). (B) IP samples were digested with PNGase F for 1h at 37°C (500 units) prior to immunoblotting for NTCP. (C) NTCP was quantified by image J and expressed relative to NTCP-WT. Molecular mass is given in kDa on the left-hand side. Results are mean +/- sd. *Indicates significant different from NTCP-WT (p <0.05).</p

Effect of NTCP glycosylation on bile acid transport activity.

<p>Taurocholate (TCA) uptake assay in parental HepG2 cells and those stably expressing NTCP-WT, NTCP-N5Q, NTCP-N11Q or NTCP-N5,11Q. Cells were incubated for 2 minutes with uptake buffer containing 20 mM taurocholate, spiked with [³H]-taurocholate. NTCP specific uptake capacity is defined as pmol TCA uptake/well, where the bars represent the mean +/- sd of three experiments each performed in quadruplicate. *Significantly different from parental cells and # indicates significantly different from NTCP-WT values (p < 0.05).</p

Overnight resting heightens sensitivity to antigens.

<p>(A) Bar charts indicate frequencies of bi- and mono-functional (blue: IFN-γ+/MIP-1β+; grey: MIP-1β+) HIV Nef specific CD8 T cells for a given peptide concentration (0.125–4 µg/ml) as percentage of total CD8 T cells. Cells were tested without (upper panel) or with (lower panel) overnight resting. (B) Pie charts show the functional profiles of HIV Nef specific CD8 T cells for a given peptide concentration. Cells were tested without (upper panel) or with (lower panel) overnight resting. (A) and (B) one representative experiment is shown. (C) Relative mean fluorescent intensity (rMFI) of IFN-γ staining of HIV Nef specific CD8 T cells of eight individuals normalized to rMFI of total CD3 T cells. All analyses were performed on not rested and rested PBMC as indicated. (** p<0.005, Wilcoxon matched pairs test; median indicated by black line).</p

Overnight resting reduces post-thaw viability of PBMC.

<p>(A) Flow cytometric analysis of lymphocyte viability compared between not rested and rested cells in a cohort of 21 individuals (** p<0.005, Wilcoxon matched pairs test; median indicated by black line). (B) Flow cytometric live/dead (NIR-/NIR+) discrimination and costaining with Annexin V for resolution between dead (NIR+) and apoptotic (NIR-/Annexin V+) cells in not rested and rested PBMC. NIR: near-infrared.</p

Antibodies used for ICS of PBMC.

<p>Antibodies used for ICS of PBMC.</p

Resting effect is not mediated by antigen presenting cells.

<p>(A) Autologous EBV-transformed B-lymphoblastoid cell lines were used as antigen presenting cells to stimulate antigen specific T cells of an EBV seropositive subject. PBMC without the addition of EBV-transformed B-lymphoblastoid cell lines provided the negative controls used for background subtraction. (B) CD3 T cells of a CMV seropositive subject were isolated by magnetic cell sorting. After cryopreservation, CD3 T cells were stimulated directly or after overnight rest with a pool of overlapping peptides corresponding to the CMV-IE-1 protein. (A) and (B) IFNγ, IL2 and MIP1β production was determined by ICS directly or after an overnight resting. Frequencies (bar charts) and functional composition (pie charts) of EBV (A) and CMV (B) specific CD8 T cells are shown. CD8 T-cell subpopulations are depicted according to their functionality (three functions: green; two functions: blue; monofunctional cells: grey). One representative experiment is shown.</p