Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: HTLV-1 infection and neuropathogenesis in the context of Rag1 −/− γc −/− (RAG1-hu) and BLT mice

doi: 10.1007/s11481-017-9740-y

Figure Lengend Snippet: (A) CD45+ CD3+ CD4+ human leukocyte expression data in peripheral blood of Rag1 and BLT hu-mice analyzed by flow cytometry. (B) Tissue sections of spleen from Rag1 hu-mice were stained for the presence of human leukocytes lightly counterstained with hematoxylin to enable visualization of nuclei. CD11b+ monocytes/dendritic cells (left) CD3+ T cells (middle), and CD20+ B cells (right) were detected in the spleen of Rag1 hu- mice.

Article Snippet: Antibodies Mouse monoclonal anti-Tax antibody, rabbit monoclonal anti-human CD4 antibody (Abcam, Cambridge, MA), rabbit polyclonal pre-diluted anti- human CD3 (Invitrogen, Carlsbad, CA) and mouse anti-human CD3 monoclonal antibodies (mAbs) (Novus Biologicals, Littleton CO), goat polyclonal anti-human PD-1 antibody (Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-human CD20 antibody (Thermo Scientific, Grand Island, NY), mouse monoclonal anti-human CD11b (eBioscience), mouse anti-human TIGIT mAb (ebioscience, San Diego, CA), mouse anti-human Tim-3 mAb (Biolegend, San Diego, CA) and rabbit monoclonal anti-human CD8 (Thermo Fisher Scientific, Waltham, MA) were used for fluorescent immunohistochemistry analyses.

Techniques: Expressing, Flow Cytometry, Staining

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: HTLV-1 infection and neuropathogenesis in the context of Rag1 −/− γc −/− (RAG1-hu) and BLT mice

doi: 10.1007/s11481-017-9740-y

Figure Lengend Snippet: At 4.4 and 10 weeks post-infection Rag1 and BLT mice were euthanized followed by the collection of brain and spinal cord. (A) Immunofluorescence revealed the presence of human CD45+ cells (green arrow) in infected hu-mice within the spinal cord. (B) Immunofluorescence imaging showed CD3+ cells and Tax expression in the spinal cord of infected hu-mice (C) CD45 cells and Tax speckles in Rag1 and BLT hu-mice were quantified using NIH Image J software and depicted as bar diagram. Error bars represent standard error of mean. Data is representative of 12 Rag1 and 10 BLT hu-mice.

Article Snippet: Antibodies Mouse monoclonal anti-Tax antibody, rabbit monoclonal anti-human CD4 antibody (Abcam, Cambridge, MA), rabbit polyclonal pre-diluted anti- human CD3 (Invitrogen, Carlsbad, CA) and mouse anti-human CD3 monoclonal antibodies (mAbs) (Novus Biologicals, Littleton CO), goat polyclonal anti-human PD-1 antibody (Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-human CD20 antibody (Thermo Scientific, Grand Island, NY), mouse monoclonal anti-human CD11b (eBioscience), mouse anti-human TIGIT mAb (ebioscience, San Diego, CA), mouse anti-human Tim-3 mAb (Biolegend, San Diego, CA) and rabbit monoclonal anti-human CD8 (Thermo Fisher Scientific, Waltham, MA) were used for fluorescent immunohistochemistry analyses.

Techniques: Infection, Immunofluorescence, Imaging, Expressing, Software

Journal: Journal of Biological Chemistry

Article Title: T Cell Receptor (TCR)-induced Tyrosine Phosphorylation Dynamics Identifies THEMIS as a New TCR Signalosome Component

doi: 10.1074/jbc.m110.201236

Figure Lengend Snippet: FIGURE 1. Quantitative analysis of TCR-dependent protein tyrosine phosphorylation. A, experimental workflow is shown. Jurkat cells were metaboli- cally labeled with three combinations of arginine and lysine containing light (R0, K0) or heavy (R6, K4 or R10, K8) isotopes until complete incorporation (as de- scribed under “Experimental Procedures”). Equal numbers of cells for each labeling condition were divided into two series (1 and 2), activated with anti-CD3 Ab at 37 °C for the indicated times, and immediately lysed in ice-cold lysis buffer. Post-nuclear lysates of each activation series were mixed in a 1:1:1 ratio and subjected to Tyr(P) (pY) immunoprecipitation for 60–90 min at 4 °C using a mixture of three anti-Tyr(P) Abs coupled to beads (4G10, Tyr(P)-99, Tyr(P)- 20). Beads were washed and eluted with phenyl phosphate, and eluates were separated by SDS-PAGE. 10 slices per gel lane were cut and digested with trypsin overnight, and the resulting tryptic peptides were analyzed by LC-MS/MS. After protein identification and quantitation, relative protein abundance in each activation series were bridged and normalized using quantitation of the common time point (0.5 min), thus, resulting in a continuous activation profile over 5 time points. B, 758 proteins were confidently identified from three independent SILAC experiments. 141 proteins that showed a significant increase (1.6-fold) above basal levels in anti-Tyr(P) immunoisolates after TCR stimulation were selected, of which 77 were retained after further manual validation. Numbers within bars indicate tyrosine-phosphorylated proteins identified in each set. C, over-represented domains within identified protein se- quences from the three SILAC experiments; most domains are involved in signaling events. ArfGAP, GTPase activating proteins toward Arf; CH, calponin ho- mology domain; RRM, RNA recognition motif; C2, calcium-dependent phospholipid binding domain; RhoGAP, Rho GTPase activating protein domain; Rho- GEF, guanine nucleotide exchange factor for Rho GTPases domain; PH, Pleckstrin homology domain; SH2, Src homology domain 2; SH3, Src homology domain 3. D, analysis of activated signaling pathways by cross-correlation with the signaling pathways data base Kegg is shown.

Article Snippet: Mouse monoclonal antibodies (Abs) used included: antiphosphotyrosine (Tyr(P)) (4G10, Millipore, PY99, Santa Cruz Biotechnology, and PY20, BD Transduction Laboratories); anti-LCK (lymphocyte-specific kinase; 3A5, Santa Cruz); antiZAP-70 (2F3.2), anti-LAT (2E9), anti-phospho-LAT (Tyr226) (Millipore); anti-CD3 (Tyr142, BD Biosciences); anti-phospho-ERK1/2 (E10, Cell Signaling Technology); anti-GAPDH (6C5, Calbiochem); anti-One-STrEP-Tag mAb (StrepMAB Classic, IBA bioTAGnology); anti-human CD3 (UCHT-1) and anti-SLP-76 (SLP-76/03, AbD Serotec); anti-human CD28 (CD28.2, BioLegend).

Techniques: Phospho-proteomics, Labeling, Lysis, Activation Assay, Immunoprecipitation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Quantitative Proteomics, Multiplex sample analysis, Biomarker Discovery, Binding Assay, Protein-Protein interactions

Journal: Journal of Biological Chemistry

Article Title: T Cell Receptor (TCR)-induced Tyrosine Phosphorylation Dynamics Identifies THEMIS as a New TCR Signalosome Component

doi: 10.1074/jbc.m110.201236

Figure Lengend Snippet: FIGURE 4. Themis is a new component of the SLP-76LAT signalosome. A, shown is a THEMIS-One STrEP tag (OST) pulldown assay using Streptactin-Sep- harose after anti-CD3 stimulation in Jurkat cells transfected with non-targeting control shRNA or a shRNA construct targeting LCK (70% knockdown effi- ciency). IB, immunoblot; pY, Tyr(P). B, THEMIS IP from anti-CD3 stimulated LAT-deficient (J.CaM2.5) and reconstituted cells is shown. C, THEMIS IP from anti- CD3 stimulated SLP-76-deficient (J14) and reconstituted cells is shown. D, THEMIS IPs from resting or anti-CD3-stimulated Jurkat cells is shown. Specific THEMIS antibody saturated with the peptide against which the antibody was raised served as a control. E, a THEMIS-OST pulldown assay from resting or CD3-stimulated Jurkat cells stably expressing THEMIS-OST using biotin-saturated Streptactin-Sepharose as control is shown. Both sets of blots were probed with antibodies against TCR-proximal signaling proteins.

Article Snippet: Mouse monoclonal antibodies (Abs) used included: antiphosphotyrosine (Tyr(P)) (4G10, Millipore, PY99, Santa Cruz Biotechnology, and PY20, BD Transduction Laboratories); anti-LCK (lymphocyte-specific kinase; 3A5, Santa Cruz); antiZAP-70 (2F3.2), anti-LAT (2E9), anti-phospho-LAT (Tyr226) (Millipore); anti-CD3 (Tyr142, BD Biosciences); anti-phospho-ERK1/2 (E10, Cell Signaling Technology); anti-GAPDH (6C5, Calbiochem); anti-One-STrEP-Tag mAb (StrepMAB Classic, IBA bioTAGnology); anti-human CD3 (UCHT-1) and anti-SLP-76 (SLP-76/03, AbD Serotec); anti-human CD28 (CD28.2, BioLegend).

Techniques: Strep-tag, Transfection, Control, shRNA, Construct, Knockdown, Western Blot, Stable Transfection, Expressing

Journal: Journal of Biological Chemistry

Article Title: T Cell Receptor (TCR)-induced Tyrosine Phosphorylation Dynamics Identifies THEMIS as a New TCR Signalosome Component

doi: 10.1074/jbc.m110.201236

Figure Lengend Snippet: FIGURE 5. THEMIS is a positive regulator of TCR-induced signaling. A, IL-2 ELISAs of shControl, shTHEMIS1, shTHEMIS2, and shLAT Jurkat cells stimulated with plate-bound anti-CD3 and soluble anti-CD28 (left panel) or staphylococcal enterotoxin E-pulsed Raji B cells (right panel) for 24 h is shown. Immunoblot (IB) analysis of the cell lines used (lower panel, shTHEMIS1 (60% knockdown), shTHEMIS2 (90% knockdown), shLAT (60% knockdown), and the non-targeting shControl). B, IL-2-luciferase assay of shControl and shTHEMIS2 (90% knockdown) is shown. IL-2-luciferase cells were transduced with either empty vector or an shRNA-resistant mutant of THEMIS (right panel) and stimulated with staphylococcal enterotoxin E-pulsed Raji B cells (left panel). C, TCR stimulation-in- duced IL-2 secretion in peripheral CD4CD25 T cells from wild-type and Themis knock-out mice is shown. Conventional CD4 cells were purified by nega- tive selection and stimulated with plate-bound anti-CD3 and soluble anti-CD28 Ab for 48 h. IL-2 concentrations in supernatants were measured by ELISA. Shown are data from three mice of each group, p 0.03.

Article Snippet: Mouse monoclonal antibodies (Abs) used included: antiphosphotyrosine (Tyr(P)) (4G10, Millipore, PY99, Santa Cruz Biotechnology, and PY20, BD Transduction Laboratories); anti-LCK (lymphocyte-specific kinase; 3A5, Santa Cruz); antiZAP-70 (2F3.2), anti-LAT (2E9), anti-phospho-LAT (Tyr226) (Millipore); anti-CD3 (Tyr142, BD Biosciences); anti-phospho-ERK1/2 (E10, Cell Signaling Technology); anti-GAPDH (6C5, Calbiochem); anti-One-STrEP-Tag mAb (StrepMAB Classic, IBA bioTAGnology); anti-human CD3 (UCHT-1) and anti-SLP-76 (SLP-76/03, AbD Serotec); anti-human CD28 (CD28.2, BioLegend).

Techniques: Western Blot, Knockdown, Luciferase, Transduction, Plasmid Preparation, shRNA, Mutagenesis, Knock-Out, Purification, Selection, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: T Cell Receptor (TCR)-induced Tyrosine Phosphorylation Dynamics Identifies THEMIS as a New TCR Signalosome Component

doi: 10.1074/jbc.m110.201236

Figure Lengend Snippet: FIGURE 6. THEMIS positively modulates NFAT/AP-1 and ERK activity. A, shown is an NFAT/AP-1-luciferase assay of shControl, shTHEMIS1 (60% knockdown), and shTHEMIS2 (90% knockdown) NFAT/AP-1-luciferase Jurkat cells after stimulation with plate-bound anti-CD3 and soluble anti-CD28 (left panel) and immunoblot (IB) analysis of THEMIS expression in the cell lines used (right panel). RLU, relative light units. B, an NF-B-luciferase assay of shControl, shTHEMIS1, and shTHEMIS2 Jurkat cells stimulated as in A (for THEMIS expression levels in cell lines used see Fig. 5A, lower panel). C, im- munoblots of ERK1/2 phosphorylation kinetics in shControl and shTHEMIS2 (90% knockdown) Jurkat cells in three independent experiments using de- creasing anti-CD3 concentrations (total ERK1/2 and ZAP-70 blots are shown as loading controls) are shown.

Article Snippet: Mouse monoclonal antibodies (Abs) used included: antiphosphotyrosine (Tyr(P)) (4G10, Millipore, PY99, Santa Cruz Biotechnology, and PY20, BD Transduction Laboratories); anti-LCK (lymphocyte-specific kinase; 3A5, Santa Cruz); antiZAP-70 (2F3.2), anti-LAT (2E9), anti-phospho-LAT (Tyr226) (Millipore); anti-CD3 (Tyr142, BD Biosciences); anti-phospho-ERK1/2 (E10, Cell Signaling Technology); anti-GAPDH (6C5, Calbiochem); anti-One-STrEP-Tag mAb (StrepMAB Classic, IBA bioTAGnology); anti-human CD3 (UCHT-1) and anti-SLP-76 (SLP-76/03, AbD Serotec); anti-human CD28 (CD28.2, BioLegend).

Techniques: Activity Assay, Luciferase, Knockdown, Western Blot, Expressing, Phospho-proteomics

Journal: Neuro-Oncology

Article Title: NEO100 enables brain delivery of blood‒brain barrier impermeable therapeutics

doi: 10.1093/neuonc/noaa206

Figure Lengend Snippet: IA NEO100 mediates human CAR T-cell penetration into brain tumors. (A) Immunostaining was performed to identify the spreading of CD3+ cells within tumor tissues harvested from the mice bearing intracranial human B-cell Raji CNS xenografts and treated with i.v. CAR-T (5 × 106 ) cells with or without IA 0.3% NEO100. Untreated animals served as a negative control, while CD3+ cells from cultures of Lym-1 CAR T cells served as positive control. IA NEO100 enabled highly preferential accumulation of CD 3+ Lym-1 CAR T cells in the lymphoma xenografts (****P < 0.0001), compared with untreated mice, or with mice treated with i.v. CAR T cells infusion alone. (B) Schematic representation showing the most relevant structural elements of the BBB, ie, ECs, pericytes, and astrocytic end-foot processes, and the relative size of, immunoglobins, white blood cells (neutrophils, 10–12 μm in diameter; and lymphocytes, 6–14 μm in diameter), and red blood cells (7.5 μm in diameter).

Article Snippet: 11 Primary antibodies used were rabbit anti-human CD3 (Cell Signaling, 99940), rabbit anti-CD8 (Abcam, ab4055), FITC-conjugated Armenian hamster anti-mouse PD-1 (ThermoFisher Scientific, 11-9985-82), and rat anti-mouse PD-1 (Bio X Cell, BE0146).

Techniques: Immunostaining, Negative Control, Positive Control