Journal: PLoS ONE

Article Title: IFN-α Is Constitutively Expressed in the Human Thymus, but Not in Peripheral Lymphoid Organs

doi: 10.1371/journal.pone.0024252

Figure Lengend Snippet: A,B , Total thymocytes were isolated from fetal and postnatal thymus tissues. Cells were stained for surface expression of CD3, CD4, CD8 and intracellular expression of MxA ex vivo . Three fetal and 18 postnatal thymus tissues were examined to determine the effect of age (A) and sex (B) on MxA expression. C , Cells from postnatal thymus, fetal thymus spleen and lymph node tissues and adult PBMC were stained for intracellular expression of MxA. Fetal tissues were obtained from the same donor.

Article Snippet: mAbs CD1a, CD3, CD4, CD8, CD27, CD45RA, CD123, and STAT1 (pY701); isotype controls IgG1 and IgG2 conjugated with FITC, PE, PerCP, PE-Cy7 or APC; and goat anti-mouse FITC were obtained from Becton Dickinson (BD); CD1a conjugated with PE from Beckman Coulter Immunotech; BDCA4-APC from Miltenyi Biotec; CD8-APC-Alexa750, CD45RA-PerCP-Cy5.5, CD1a-Pacific Blue and CD3-650 eFluor (Nano-crystals) from eBioscience; IFN-α -biotin (Endogen); isotype control IgG2a pure, Goat anti-Rabbit PE from Caltag; mAb to CD27 from Lab Vision; Goat anti-mouse IgG1 AlexaFluor-488, Goat anti-mouse IgG2a AlexaFluor-594, Goat anti-Rabbit AlexaFluor-594, and Zenon anti IgG2a AlexaFluor-488 from Molecular Probes. mAb to MxA was generously provided by Drs. Haller and Kochs (University of Freiburg, Germany). mAb CD1a pure, TBST, Antibody Diluent, and Target Retrieval Solution were obtained from DAKO (Carpinteria, CA); rabbit anti-mouse IgG1 pure from Zymed; mAb IRF-7 from Santa Cruz Biotechnology; mAb LL-37 from Cell Sciences.

Techniques: Isolation, Staining, Expressing, Ex Vivo

Journal: PLoS ONE

Article Title: IFN-α Is Constitutively Expressed in the Human Thymus, but Not in Peripheral Lymphoid Organs

doi: 10.1371/journal.pone.0024252

Figure Lengend Snippet: A, B, Total thymocytes were stained for surface expression of CD4, CD8, CD1a, CD3, CD27, CD45RA and CD123 combined with intracellular MxA expression. A, Differences of thymocyte subset expression patterns in all thymocytes, MxA negative and positive cells are shown. B , Differences of thymocyte subset expression patterns in CD27 + , MxA negative and positive cells are shown. C, Total thymocytes were cultured in the presence or absence of 1000 U/mL IFN-α overnight. Cells were stained for surface expression of CD4, CD8, CD1a, CD3, CD27, CD45RA and CD123 combined with intracellular MxA expression. MxA expression in immature and mature thymocyte subsets was determined by gating on CD1a + CD27 − and CD1a − CD27 + cells respectively.

Article Snippet: mAbs CD1a, CD3, CD4, CD8, CD27, CD45RA, CD123, and STAT1 (pY701); isotype controls IgG1 and IgG2 conjugated with FITC, PE, PerCP, PE-Cy7 or APC; and goat anti-mouse FITC were obtained from Becton Dickinson (BD); CD1a conjugated with PE from Beckman Coulter Immunotech; BDCA4-APC from Miltenyi Biotec; CD8-APC-Alexa750, CD45RA-PerCP-Cy5.5, CD1a-Pacific Blue and CD3-650 eFluor (Nano-crystals) from eBioscience; IFN-α -biotin (Endogen); isotype control IgG2a pure, Goat anti-Rabbit PE from Caltag; mAb to CD27 from Lab Vision; Goat anti-mouse IgG1 AlexaFluor-488, Goat anti-mouse IgG2a AlexaFluor-594, Goat anti-Rabbit AlexaFluor-594, and Zenon anti IgG2a AlexaFluor-488 from Molecular Probes. mAb to MxA was generously provided by Drs. Haller and Kochs (University of Freiburg, Germany). mAb CD1a pure, TBST, Antibody Diluent, and Target Retrieval Solution were obtained from DAKO (Carpinteria, CA); rabbit anti-mouse IgG1 pure from Zymed; mAb IRF-7 from Santa Cruz Biotechnology; mAb LL-37 from Cell Sciences.

Techniques: Staining, Expressing, Cell Culture

Journal: PLoS ONE

Article Title: IFN-α Is Constitutively Expressed in the Human Thymus, but Not in Peripheral Lymphoid Organs

doi: 10.1371/journal.pone.0024252

Figure Lengend Snippet: A , pDC from post-natal thymus express IFN-α. Total thymocytes were stained for surface expression of CD123, CD45RA, CD4, CD8, CD27, CD1a and CD3 and intracellular expression of IFN-α. IFN-α expression of pDC was determined by gating on CD45RA + CD123 +/hi cells, that lacked CD3 and CD1a. B , pDC from post-natal thymus express MxA and pSTAT1. Total thymocytes were stained for surface expression of CD45RA, CD123, and CD3 and intracellular expression of MxA or pSTAT1. MxA and pSTAT1 expression in pDC was determined by gating on CD45RA + CD123 +/hi cells that lacked CD3. C , pDC express the highest level of MxA. Total thymocytes were stained for surface expression of CD45RA, CD123, and CD3 and intracellular expression of MxA. Mean fluorescence intensity (MFI) of MxA expression in pDC was determined by gating on CD3 − CD45RA + CD123 +/hi cells. MxA MFI expression in mature and immature thymocyte subsets was determined by gating on CD45RA + CD3 +/hi , CD45RA − CD3 + and CD3 low/− cells. D, pDC are located in the medulla of normal thymus. Thymus tissue was formalin fixed and paraffin embedded. 4 um sections were mounted on slides. Thymus tissue was stained for CD123 followed by Rabbit anti-mouse IgG1 and Goat anti-Rabbit Alexa Fluor 594. MxA was conjugated with zenon anti-IgG2a Alexa 488.

Article Snippet: mAbs CD1a, CD3, CD4, CD8, CD27, CD45RA, CD123, and STAT1 (pY701); isotype controls IgG1 and IgG2 conjugated with FITC, PE, PerCP, PE-Cy7 or APC; and goat anti-mouse FITC were obtained from Becton Dickinson (BD); CD1a conjugated with PE from Beckman Coulter Immunotech; BDCA4-APC from Miltenyi Biotec; CD8-APC-Alexa750, CD45RA-PerCP-Cy5.5, CD1a-Pacific Blue and CD3-650 eFluor (Nano-crystals) from eBioscience; IFN-α -biotin (Endogen); isotype control IgG2a pure, Goat anti-Rabbit PE from Caltag; mAb to CD27 from Lab Vision; Goat anti-mouse IgG1 AlexaFluor-488, Goat anti-mouse IgG2a AlexaFluor-594, Goat anti-Rabbit AlexaFluor-594, and Zenon anti IgG2a AlexaFluor-488 from Molecular Probes. mAb to MxA was generously provided by Drs. Haller and Kochs (University of Freiburg, Germany). mAb CD1a pure, TBST, Antibody Diluent, and Target Retrieval Solution were obtained from DAKO (Carpinteria, CA); rabbit anti-mouse IgG1 pure from Zymed; mAb IRF-7 from Santa Cruz Biotechnology; mAb LL-37 from Cell Sciences.

Techniques: Staining, Expressing, Fluorescence

Journal: Therapeutic Advances in Medical Oncology

Article Title: Exceptional response to nivolumab of a heavily pre-treated patient with metastatic renal-cell cancer: from a case report to molecular investigation and future perspectives

doi: 10.1177/1758835920946152

Figure Lengend Snippet: Photomicrographs of FFPE tissue from primary and metastatic lesions stained for PD-L1, CD3, CD8, CD20, CD138, CD1a, and FoxP3 and assessed by immunohistochemistry in primary and metastatic lesions.

Article Snippet: The following primary antibodies were employed: anti-CD3 rabbit polyclonal Ab (DAKO, CA, USA), diluted 1:300; anti-CD8 mouse monoclonal Ab (mAb), clone C8/144B (DAKO, CA, USA), ready to use; anti-Foxp3 mouse mAb, clone 20034 (Abcam, Cambridge, UK), diluted 1:100; anti-CD20 mouse mAb, clone L26 (DAKO, CA, USA), diluted 1:200; anti-CD138 mouse mAb, clone MI15 (DAKO, CA, USA), diluted 1:100; anti-CD1a mouse mAb, clone 010 (DAKO, CA, USA), ready to use; MLH1 mouse mAb, clone ES05 (DAKO, CA, USA), ready to use; MSH2 mouse mAb, clone FE11 (DAKO, CA, USA), ready to use; MSH6 rabbit mAb, clone EP49 (DAKO, CA, USA), ready to use; and PMS2 rabbit mAb, clone EP51 (DAKO, CA, USA), ready to use.

Techniques: Staining, Immunohistochemistry

Journal: European journal of immunology

Article Title: Contact sensitizers trigger human CD1-autoreactive T-cell responses

doi: 10.1002/eji.201746939

Figure Lengend Snippet: DNCB-pulsed CD1+APCs trigger the activation of CD1a- and CD1d-restricted clones. (A-D) B13 cells (A, B) and S33d cells (C, D) were stimulated with (A) DCs, (B) THP-1 CD1a, and (C) THP-1 CD1d cells by pulsing for 24 h with DNCB. (D) S33d cells were stimulated with sulfatide presented by THP-1 CD1d cells previously pulsed with DNCB (6 μM, open circles), or DMSO vehicle (VEH, closed circles). Production of (A, B) GM-CSF and (C, D) IFN-γ was measured by ELISA and shown as mean ± SD, n=3 for B13, n=4 for S33d cells. *p 0.01, t-test with Sidak multiple comparisons. Data shown are from single experiments representative of 3 independent experiments. (E) Heatmap of cytokines produced by B13 cells cultured with THP-1 CD1a cells previously pulsed with DNCB (6 μM) or VEH. Normalized data is expressed as the z-score. Absolute cytokine values are illustrated in Supporting Information Fig. 2. The following cytokines were tested, but were not released by the T-cell clone: IL-15, IL-17a/e/f, IL-21, IL-23, IL-27, IL-28, IL-33, TNF-β and MIP-1α. (F) Heatmap of cytokines produced by S33d cells cultured with THP-1 CD1d cells previously pulsed with DNCB (6 μM) or VEH. Normalized data are expressed as the z-score. Absolute cytokine values are illustrated in Supporting Information Fig. 3. The following cytokines were tested, but were not released by the T-cell clone: IL-5, IL-6, IL-9, IL-15, IL-17a/e/f, IL-21, IL-22, IL-23, IL-27, IL-28, IL-31, IL-33, TNF-β and MIP-1α. (E, F) Data in each column represent an individual replica of the indicated experimental condition.

Article Snippet: Human CD1 molecules were detected with mAbs specific for CD1a (HI149), CD1b (SN13), CD1c (L161) and CD1d (51.1, all eBioscience), following pre-incubation with Fc-block (BD Pharmingen).

Techniques: Activation Assay, Clone Assay, Enzyme-linked Immunosorbent Assay, Produced, Cell Culture

Journal: European journal of immunology

Article Title: Contact sensitizers trigger human CD1-autoreactive T-cell responses

doi: 10.1002/eji.201746939

Figure Lengend Snippet: DNCB activity is mediated by CD1 and TCR. (A, B) Anti-CD1a (α-CD1a, A) and anti-CD1d (α-CD1d, B) or isotype-matched control mAbs (IgG1 and IgG2b, respectively) were added to (A) THP-1 CD1a cells and (B) THP-1 CD1d cells pulsed with DNCB (6 μM) or DMSO vehicle, before the incubation with (A) B13 and (B) S33d cells. Cytokines released by the T cells are expressed in ng/ml (mean + SD, n= 3-4) and results are from single experiments representative of 3 independent experiments. *p ≤0.001 (t-test with Sidak multiple comparisons). (C, D) Flow cytometry analysis of CD69 surface expression by (C) SKW-B13 and (D) SKW-S33d cells following incubation with (C) THP-1 CD1a and (D) THP-1 CD1d cells pretreated with DNCB. Control CD3-SKW-3 cells are also shown. Median fluorescence intensity (MFI) of CD3+ and of CD3- cells is plotted. Each plot represents data from a single experiment and is representative of 2 independent experiments.

Article Snippet: Human CD1 molecules were detected with mAbs specific for CD1a (HI149), CD1b (SN13), CD1c (L161) and CD1d (51.1, all eBioscience), following pre-incubation with Fc-block (BD Pharmingen).

Techniques: Activity Assay, Control, Incubation, Flow Cytometry, Expressing, Fluorescence

Journal: European journal of immunology

Article Title: Contact sensitizers trigger human CD1-autoreactive T-cell responses

doi: 10.1002/eji.201746939

Figure Lengend Snippet: DNCB potentiates S33d cell activation through endogenous lipids. (A) IFN-γ response of S33d T cells to sulfatide presented by C1R CD1d cells pulsed with DNCB (6 μM, open circles) or DMSO vehicle (VEH, closed circles). (B) Non-stimulatory d18:1 C22:1 sulfatide was added to displace endogenous lipids from THP-1 CD1d cells previously pulsed with DNCB (6μM, open circles) or VEH (closed circles), before assessing S33d T-cell response. (C) Control response of the CD1a-restricted T-cell clone K34B9.1 to d18:1 C22:1 sulfatide presented by THP-1 CD1a cells. Data are expressed as mean ± SD, n=4,. *p≤0.05, t-test with Sidak multiple comparisons. Data are from single experiments representative of 2-3 independent experiments.

Article Snippet: Human CD1 molecules were detected with mAbs specific for CD1a (HI149), CD1b (SN13), CD1c (L161) and CD1d (51.1, all eBioscience), following pre-incubation with Fc-block (BD Pharmingen).

Techniques: Activation Assay, Control

Journal: European journal of immunology

Article Title: Contact sensitizers trigger human CD1-autoreactive T-cell responses

doi: 10.1002/eji.201746939

Figure Lengend Snippet: A wide range of contact sensitizers induce CD1-mediated T-cell activation. (A-C) The IFN-γ response of S33d cells to THP-1 CD1d cells pulsed with (A) resorcinol, (B) isoeugenol and (C) cinnamaldehyde. (D) S33d cell response in the presence of anti-CD1d (α-CD1d) or irrelevant (IgG2a) mAbs. Resorcinol was solubilized in medium and used at 2 mM, isoeugenol was solubilized in DMSO and used at 250 μM, cinnamaldehyde was solubilized in DMSO and used at 40 μM. (E) Response of B13 cells to THP-1 CD1a cells pulsed with 1,4 benzoquinone (10 μM in medium) and in the presence of anti-CD1a or irrelevant (IgG1) mAbs (α-CD1a). Data are expressed as mean ± SD, n=2-3, and are from single experiments representative of 2-3 independent experiments. *p 0.01 vs. the relevant vehicle controls, t-test with Sidak multiple comparisons.

Article Snippet: Human CD1 molecules were detected with mAbs specific for CD1a (HI149), CD1b (SN13), CD1c (L161) and CD1d (51.1, all eBioscience), following pre-incubation with Fc-block (BD Pharmingen).

Techniques: Activation Assay