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)

FACS analysis with sera specific for well-known proteins.

<p>(A) Comparison of the CD8 staining performed on PBL with either a commercially available anti CD8 mAb (BD biosciences) or the anti CD8 alpha antiserum at 1∶100 dilution points. Both the samples were stained also with commercially available anti CD3 and anti CD4 mAb (BD biosciences). The distribution of CD4 and CD8 is analyzed upon gating on CD3 positive cells. (B) Examples of staining with antisera from the library. PBLs from healthy donors were stained with anti CD2, CD1d, CD8 alpha, CD25, CD72, CD80, CD38, and CD86. The expression of CD25 was assessed upon a 24 hours activation of PBLs with 1 µg/ml of PHA. The expression of CD80 and CD86 was assessed upon gating on monocytes after a 24 hours activation of PBLs with 1 µg/ml of PHA. The expression of CD133 was analyzed on cord blood derived CD34+, CD45dim cells. Serum from not immunized mice was used as negative control in all the stainings.</p

Schematic representation of the antisera library generation.

<p>Schematic representation of the antisera library generation.</p

Comparison of the results obtained with antisera specific for well-known proteins assessed both by FACS on PBMCs from healthy donors and by IHC on inflamed lymph nodes.

<p>Comparison of the results obtained with antisera specific for well-known proteins assessed both by FACS on PBMCs from healthy donors and by IHC on inflamed lymph nodes.</p

Results of sera screening by FACS on PBL and Cord Blood cells.

<p>(A) FACS analysis of sera positive on PBLs. PBLs were stained with the indicated sera at the optimal dilution point (1∶50 to 1∶200). The samples were stained also with anti CD3, anti CD19 and anti CD56 mAbs to analyze the sera reactivity upon gating on the different subpopulations. A plot representative of five different donors is shown for each serum. (B) KRTCAP-3 specific serum recognizes PHA-treated cells. PBMCs are treated for 24 hours with 1 µg/ml of PHA. After the treatment both un-stimulated and treated cells are stained with the KRTCAP-3-specific serum. (C) FACS analysis of sera positive on cord blood cells. Cord blood mononuclear cells are stained with the indicated sera at the optimal concentration (1∶50 to 1∶100). The samples are stained also with anti CD45 and anti CD34 mAbs to perform the analysis upon gating on CD34highCD45dim. A plot representative of a least 3 independent donors is shown. Il all the cases (A,B,C,) a staining with the serum of not immunized mice was used as negative control. (D) RT-PCR analysis. a- cDNA from total PBMC were amplified with primers specific for the indicated proteins. b- cDNA from both un-stimulated and PHA-treated PBMC was amplified with KRTCAP-3 specific primers. KRTCAP3 expression is up regulated two to three times. Beta actin amplification is used as normalization. c- cDNA samples from CD34+CD45dim cells were generated by retro-transcription of RNA extracted from a pool of CD34 positive cells from 2–3 independent cord blood units magnetically purified using the Miltenyi CD34 microbeads kit according the manufacturer instruction. The purity of the CD34+CD45dim cells was usually >99%. The samples were amplified with primers specific for the indicated proteins and described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034395#s2" target="_blank">Methods</a> section.</p

Assessment of antisera specificity on Hela transfected cells.

<p>Hela cells were transiently transfected with a myc-tag version of the proteins identified with the sera library. At 24 hours from the transfection cells were lysated as described in the Method section. 40 µg of total proteins were loaded on SDS page and a WB analysis was performed using both an anti myc mAb (9E10 clone) and the corresponding antiserum. (A) WB analysis of Hela cells transfected with CRISP-1 and MOSC-1. In both the cases the anti myc mAb and the specific antiserum recognized a protein of the expected molecular weight that is not present in the cells transfected with the mock vector. A comparable result was obtained wit KRTCAP-3 (B), TMCC-1 (C), TMEM38B (D) and SUSD3 (E) transfected cells. The WB analysis of GSG1-L cells (E) and LPPR2 cells (F) shows that neither the anti myc nor the specific antiserum is able to recognize in a specific way a protein in transfected cells.</p