Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides-1

<p><b>Copyright information:</b></p><p>Taken from "Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides"</p><p>BMC Immunology 2005;6():24-24.</p><p>Published online 5 Dec 2005</p><p>PMCID:PMC1325046.</p><p>Copyright © 2005 Moro et al; licensee BioMed Central Ltd.</p>ipitation of HLA-DR*1101-Ig and HLA-DR*1101-Bir molecule with 30 μl of L243-sepharose beads from 1 ml of S2 cells culture supernatant after CuSO4 induction. The lanes contain the following material: 1. sup, 30 μl of culture supernatant before immunoprecipitation; 2. bound, the immunoprecipitated soluble recombinant HLA-DR*1101; and 3. not bound, 30 μl of culture supernatant after immunoprecipitation. Proteins were separated on SDS-page, transferred to filter and revealed with anti his-tag antibody. (b) Densitometric analysis of the protein bands displayed in panel (a), showing the elative efficiency of immunoprecipitation of the two soluble recombinant HLA-DR*1101 proteins with L243-Sepharose beads, as an indirect indicator of the percentage of correctly folded molecule. (c) Size exclusion chromatography of HLA-DR*1101-Ig molecules, after purification of ProtA affinity chromatography. The elution profile of the molecule from a Superdex 200 gel filtration column is shown. Inset shows the dot-blot analysis performed on the protein contained in the indicated elution peaks. Spotted proteins are probed with either anti-His tag antibody, to verify the presence of the HLA-DR*1101 molecule, or L243 mAb (Anti-HLA-DR) to verify the correct conformation of the molecule. (d) Elution profile from Superdex 200 gel filtration column and dot-blot analysis on eluted peaks of HLA-DR11-Bir molecules, performed as described in (c). (e) Calibration profile of the Superdex 200 gel filtration column

Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides-4

<p><b>Copyright information:</b></p><p>Taken from "Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides"</p><p>BMC Immunology 2005;6():24-24.</p><p>Published online 5 Dec 2005</p><p>PMCID:PMC1325046.</p><p>Copyright © 2005 Moro et al; licensee BioMed Central Ltd.</p>lation with p2 and APCs. The histograms in the upper row display the amount of surface TCR (mfi) expressed by the T cells at the time of tetramer staining, as determined by anti-CD3 staining. The histograms in the lower row show the staining with the p2-loaded HLA-DR*1101-Bir tetramers (mfi) performed at the indicated time after restimulation

Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides-3

<p><b>Copyright information:</b></p><p>Taken from "Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides"</p><p>BMC Immunology 2005;6():24-24.</p><p>Published online 5 Dec 2005</p><p>PMCID:PMC1325046.</p><p>Copyright © 2005 Moro et al; licensee BioMed Central Ltd.</p>aded HLA-DR*1101-Bir tetramers at different temperature. (a) The relative affinity for the p2-HLA-DR*1101 displayed by the two TCC clones 162 and 51 is determined in an IFN-γ-releasing assay following production in response to different doses of p2 peptide. 10T cells are incubated with 5 × 10HLA-DR*1101 LCL cells and the indicated doses of p2 peptide. After 48 hours, the concentration of IFN-γ in the culture supernatant is measured by ELISA. Indicated are the concentration of p2 peptide required to elicit half-maximum release of IFN-γ in the two TCC. (b) Staining of TCC162, TCC51 and the irrelevant TCC with either p2-loaded HLA-DR*1101-Ig or p2-loaded HLA-DR*1101-Bir tetramers. Staining is performed for 2 hours at the indicated temperatures with 10 μg of tetramer per sample. The tetramer staining on CD3CD4gated cells is shown. The amount of surface TCR, obtained by staining with anti-CD3 mAb, expressed by the TCC in the different conditions is shown by the mean fluorescent intensity (CD3 mfi)

Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides-6

<p><b>Copyright information:</b></p><p>Taken from "Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides"</p><p>BMC Immunology 2005;6():24-24.</p><p>Published online 5 Dec 2005</p><p>PMCID:PMC1325046.</p><p>Copyright © 2005 Moro et al; licensee BioMed Central Ltd.</p>roduction to different doses of p39 peptide. 10T cells are incubated for 48 hours with 5 × 10HLA-DR*1101LCL cells and the indicated doses of p39 peptide. IFN-γ in the supernatant is measured by ELISA. (b) Staining of a MAGE-3-specific T cell clone. The cells are stained at day 19 from re-stimulation with 10 μg of either CLIP-loaded or p39-loaded HLA-DR*1101-Bir tetramers for 2 hours at 37°C

Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides-5

<p><b>Copyright information:</b></p><p>Taken from "Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides"</p><p>BMC Immunology 2005;6():24-24.</p><p>Published online 5 Dec 2005</p><p>PMCID:PMC1325046.</p><p>Copyright © 2005 Moro et al; licensee BioMed Central Ltd.</p> from a HLA-DR*1101 healthy donor after one round of stimulation with 5 μM of p2-peptide and 40 u/ml of IL-2. (b) Intracellular production of IFN-γ by T cells contained in the p2-enriched PBMC. T cells were stimulated for 6 hours with p2 in the presence of HLA-DR*1101LCL cells at 37°C. Brefeldin A was added after the first hour of stimulation. The cells were then fixed, permeabilised and stained with anti-IFN-γ mAb. Cells unstimulated (nil) and stimulated policlonally with PMA-Ionomycin (PMA/ionomycin) are shown as controls. Numbers in quadrants indicate the percentage of T cells stained with anti-IFN-γ mAb (c) Staining of PBMCs from HLA-DR*1101 healthy donor with HA-loaded HLA-DR*1101-Bir tetramers. T cells were after three rounds of stimulation with 1 μg/ml of HA peptide and 40 u/ml of IL-2. The staining is performed at the indicated days after the third stimulation. Numbers in quadrants indicate the percentage of T cells stained by HLA-DR tetramers. (d) Intracellular production of IFN-γ by CD4T cells contained in the HA-specific T cell line at day 8 from the third stimulation. Activation and staining of CD4T cells was performed as described in (b)

<i>Salmonella</i>-specific B cells form a survival niche supporting in vivo <i>Salmonella</i> spreading to systemic sites.

<p>C57BL/6 mice were adoptively transferred with 0, 2*10⁵ or 10⁶ HEL-specific CD43- naïve B cells labeled with CFSE, as indicated. Mice were orally infected with surface HEL-expressing <i>Salmonella</i> one day after B cell transfer. (A) Distribution of CFSE-labeled HEL-specific B cells in the mesenteric lymph nodes (mLN) and spleen before infection (BI), and 24 or 72 hours post-infection, as indicated. One representative example from 3 experiments with 4 mice for each experimental setting is shown. (B) Recovery of viable bacteria 72 hours post-infection from mesenteric lymph nodes (mLN) spleen (SP) and blood (BL) in infected mice transferred with 0, 2*10⁵ or 10⁶ HEL-specific B cells. Depicted are colony-forming units (CFU)/10⁵ eukaryotic cells. One representative example from 3 experiments with 4 mice for each experimental setting is shown.</p

<i>Salmonella</i> is actively excreted by B cells.

<p>(A) Primary B cells having phagocytosed anti-BCR coated GFP-<i>Salmonella</i> on a monolayer of 3T3-CD40L fibroblasts were imaged using widefield fluorescence microscopy. Depicted is the GFP signal projected on the transmission image with images taken every 30 min. Scalebar = 10 µm. Arrows indicate the B cell, white arrow: B cells moves op top of the monolayer, black arrow: B cells moves below the monolayer. Images are frames from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050667#pone.0050667.s005" target="_blank">Video S4</a>. (B) Primary B cells having phagocytosed anti-BCR coated GFP-<i>Salmonella</i> on a monolayer of 3T3-CD40L fibroblasts were imaged using widefield fluorescence microscopy in the presence of TexasRed labeled anti-LPS mAbs. Depicted are GFP and Texas-Red signals projected on the transmission image. Scalebar = 10µm. Images are frames from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050667#pone.0050667.s006" target="_blank">Video S5</a>. (C) Quantification of <i>Salmonella</i> secretion from B cells. Primary B cells were incubated with live uncoated GFP-<i>Salmonella</i>. Cells were stained with antibodies against LPS, fixed and analyzed using FACS. Left panel: increase in cell surface exposed LPS from bacteria exposed at the cell surface after initial uptake by B cells. Middle panel: percentage of B cells having excreted <i>Salmonella</i> as calculated from the percentage of B cells containing GFP-<i>Salmonella</i> followed in time. Right panel: left and middle panels are projected to illustrate that both processes show similar kinetics. Error bars represent SD from three independent experiments. (D) Primary B cells were incubated with live uncoated GFP-expressing <i>Salmonella</i> and followed for the time points indicated. The fraction of living B cells is plotted to demonstrate that loss of GFP-<i>Salmonella</i> positive B cells is not correlated with cell death.</p

Quantification of the fate of the GFP-expressing <i>Salmonella</i> in infected B cells.

<p>(A) B cells were infected with anti-BCR coated GFP-expressing <i>Salmonella</i> (green).The plasma membrane of the B cells (red) was stained using an anti-CD20 mAb to discriminate between intracellular and extracellular <i>Salmonella</i>. (B) The relative amount of intracellular <i>Salmonella</i> were measured immediately after infection (0 h), or 4 h and 18 h post-infection. Error bars represent SD from two independent experiments. (C) The number of <i>Salmonella</i> per B cell was measured immediately after infection and 18h post-infection. A representative experiment of two individual experiments is shown.</p

After excretion by B cells, <i>Salmonella</i> is capable of infecting secondary host cells.

<p>(A) Left panel: the effect of antibiotics on the growth of lux-<i>Salmonella</i> in Ramos B cells. Right panel: the same FACS analysis with primary B cells as in 2C was performed in presence of either Gentamicin or Tetracycline to discriminate between host and bacteria mediated excretion. (B) Quantification of <i>Salmonella</i> secretion from B cells. Primary B cells were incubated with live uncoated GFP-<i>Salmonella</i> in presence of either Gentamicin or Tetracycline to discriminate between host versus bacterial-mediated excretion. Cells were stained with antibodies against LPS, fixed and analyzed using FACS. Increase in cell surface LPS levels is similar in the presence of Gentamicin and Tetracycline, indicating that viable <i>Salmonella</i> are not required for excretion. (C) Primary B cells having phagocytosed anti-IgM coated GFP-<i>Salmonella</i> on a monolayer of 3T3-CD40L fibroblasts were imaged using widefield fluorescence microscopy for the times indicated. Imaging conditions are similar as in 2A. GFP-<i>Salmonella</i> is excreted from a primary B cell (white arrowhead), followed by infection of the 3T3-CD40L monolayer (outline of infected cell marked by a dashed line). Inset shows zoom-in on primary B cell excreting GFP-<i>Salmonella</i>. Images are frames from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050667#pone.0050667.s007" target="_blank">Video S6</a>.</p

BCR-mediated internalization of <i>Salmonella</i> by primary human B cells.

<p>(A) Immunocryoelectron micrograph of primary human B cells that had phagocytosed anti-BCR coated <i>Salmonella</i>. Gold particles indicate staining for CD63, black asterisks mark bacteria, N marks the nucleus and PM the plasma membrane. (B) Primary B cells were either or not pre-incubated with F(ab)₂ fragments of the anti-IgM antibody before incubation with live anti-IgM coated GFP-expressing <i>Salmonella</i>. After extensive washing, cells were fixed and analyzed by FACS indicating a strong reduction in GFP-<i>Salmonella</i> following competition with F(ab)₂ for BCR interactions. Shown is a representative plot of 5 donors tested.</p