Journal: Cellular and Molecular Immunology

Article Title: γδ T-cell autoresponses to ectopic membrane proteins: a new type of pattern recognition

doi: 10.1038/s41423-025-01258-x

Figure Lengend Snippet: Two CDR3δ peptides, GTM and OT3, with tumor binding specificity served as probes to screen the protein ligands of human γδ TCRs. a and b. Immunohistochemical staining of a tumor tissue array with the biotin-labeled GTM peptide a and biotin-labeled OT3 peptide b followed by HRP-conjugated streptavidin. The binding was visualized using DAB as the substrate (brown) (400×). Cancer: tumor tissue; paracancerous: paracarcinoma tissues. Scale bar, 50 μm. GTM and OT3 can specifically bind to several tumor cell lines. Fifty-three types of human tumor cell lines were stained with biotinylated (bio)-GTM c or (bio)-OT3 peptide d and APC-conjugated streptavidin. The binding activity was measured by flow cytometry. The flow data were standardized by the ratio between the mean fluorescence intensity of the peptide conjugate and the secondary antibody alone. The binding strength of the synthetic peptide was compared with that of normal human peripheral blood mononuclear cells (PBMCs). MST analysis of the binding activities of the GTM peptide e and OT3 peptide f to known γδ TCR-recognized stress-inducible ligands. The Kd value for each protein was calculated. g Schematic diagram of OT3- and GTM-γδ CAR molecules. A single-chain Vγ9-Vδ2 or Vγ4-Vδ1 structure, which is composed of a Vδ2 TCR with the tumor-specific CDR3δ sequence OT3 or Vδ1 TCR with the tumor-specific CDR3δ sequence GTM, forms the extracellular region of the γδ CAR. The transmembrane and intracellular segments of a classical third-generation CAR (CD8-CD28-4-1BB-CD3ζ) were linked to the Vγ-Vδ sequence. γδ CARs were cloned and inserted into the lentiviral vector cFUGW. Flow cytometry was used to assess the expression of the activation marker CD69, the secretion of TNF-α h and the cytotoxicity-related molecules granzyme B and perforin i in γδ CAR-T cells after coculture with OVCAR-8 cells in vitro. The statistical data are representative of three independent experiments and are expressed as the means ± SDs. *, p < 0.05; **, p < 0. 01. Cytotoxicity of OT3 CAR-T cells j and GTM CAR-T cells k in different tumor cells in vitro. Cytotoxicity was analyzed with an LDH cytotoxicity detection kit. The data are representative of three independent experiments and are expressed as the means ± SDs. OT3 CAR-αβ T cells exhibit strong cytotoxic activity against tumors in an NSG mouse model. OVCAR-8-transplanted NSG mice were treated with GFP-αβ T cells or OT3 CAR-αβ T cells. Tumor growth in every mouse at each time point was measured by assessing the radiance of the tumor cells l . The tumor growth curve m and survival curve n of each group are shown

Article Snippet: In the OT3 protein microarray, OT3 peptide or biotin, as the primary antibody, was diluted, added, and incubated at 4 °C for 12 h. Cy3-conjugated streptavidin (Bioss) was used to probe the primary antibodies bound to the slide at RT for 1 h. For all the assays, a rabbit anti-GST monoclonal antibody (CST) was added and incubated at RT for 1 h. An Alexa Fluor 647-conjugated goat anti-rabbit IgG (CST) secondary antibody was added and incubated at RT for 1 h. Finally, the microarray slides were scanned with a GenePix 4000B fluorescent microarray scanner (GBI).

Techniques: Binding Assay, Immunohistochemical staining, Staining, Labeling, Activity Assay, Flow Cytometry, Fluorescence, Sequencing, Clone Assay, Plasmid Preparation, Expressing, Activation Assay, Marker, In Vitro

Journal: The FASEB Journal

Article Title: Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1

doi: 10.1096/fj.11-187005

Figure Lengend Snippet: Membrane KCa3.1 is ubiquitylated during endocytic trafficking. A) At time 0 min, KCa3.1 is localized at the plasma membrane (red) and is completely endocytosed after 90 min incubation at 37°C. B) To detect ubiquitylated KCa3.1, plasma membrane channel was enzymatically biotinylated and labeled with streptavidin; cells at time 0 and 90 min were lysed in the presence of GST-TUBE2. Following pulldown, the immunoblot (IB) was probed using α-streptavidin Ab to identify BLAP-tagged KCa3.1/streptavidin. To control for nonspecific labeling, KCa3.1- expressing cells were labeled with streptavidin in the absence of enzymatic biotinylation (control lanes in all IB). No channel is detected in these controls, confirming the specificity of our labeling protocol. As shown in lane 3, endocytosed KCa3.1 is heavily ubiquitylated (time 90 min) compared with channel present at the plasma membrane (time 0 min; lane 2). Importantly, similar levels of KCa3.1 were detected for the two time points, as assessed by IB of total cell lysate (lanes 5 and 6). C) Samples were prepared and lysed as in B, then KCa3.1 was immunoprecipitated using streptavidin Ab, and the subsequent IB was probed using α-ubiquitin Ab. KCa3.1 is strongly ubiquitylated following endocytosis. D) HEK cells were doubly transfected with BLAP-KCa3.1 and HA-ubiquitin. Ubiquitin was immunoprecipitated using an anti-HA Ab and subsequently IB for streptavidin-tagged KCa3.1. E) KCa3.1 was immunoprecipitated using an anti-streptavidin antibody and the subsequent IB probed with anti-HA Ab.

Article Snippet: Antibodies Polyclonal α-streptavidin Ab was obtained from Genscript Corporation (Piscataway, NJ, USA).

Techniques: Membrane, Clinical Proteomics, Incubation, Labeling, Western Blot, Control, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Transfection

Journal: The FASEB Journal

Article Title: Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1

doi: 10.1096/fj.11-187005

Figure Lengend Snippet: Ubiquitylation is required for KCa3.1 endocytosis. A) BLAP-KCa3.1 was enzymatically biotinylated at the cell surface and labeled with streptavidin-Alexa555 or streptavidin and incubated at 37°C for 90 min in the absence or presence of hypertonic sucrose, an inhibitor of endocytosis, or the ubiquitin-activating enzyme (E1) inhibitor UBEI-41. Both hypertonic sucrose and UBEI-41 dramatically reduced the internalization of KCa3.1. B) GST-TUBE pulldown assay, followed by IB using α-streptavidin Ab, suggests that KCa3.1 may be ubiquitylated at the plasma membrane in the absence of endocytosis (compare lanes 5 and 2) and that inhibition of ubiquitylation blocks endocytosis of the channel (UBEI-41 treatment). Subsequent to endocytosis, KCa3.1 becomes heavily polyubiquitinated (T=90 min, lane 3).

Article Snippet: Antibodies Polyclonal α-streptavidin Ab was obtained from Genscript Corporation (Piscataway, NJ, USA).

Techniques: Labeling, Incubation, Ubiquitin Proteomics, Clinical Proteomics, Membrane, Inhibition

Journal: The FASEB Journal

Article Title: Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1

doi: 10.1096/fj.11-187005

Figure Lengend Snippet: KCa3.1 is deubiquitylated before delivery to lysosomes. A) BLAP-KCa3.1 was enzymatically biotinylated and streptavidin labeled; the channel was allowed to internalize for the times indicated in the absence or presence of PR-619. PR-619 treatment significantly inhibited KCa3.1 degradation (bar graph, n=3). *P < 0.05. B) Cells were treated and labeled as in A, and the ubiquitylation of KCa3.1 was assessed by GST-TUBE pulldown assay. A representative blot is shown, indicating that PR-619 treatment prevents channel deubiquitylation. These data demonstrate that KCa3.1 needs to be deubiquitylated for proper lysosomal degradation.

Article Snippet: Antibodies Polyclonal α-streptavidin Ab was obtained from Genscript Corporation (Piscataway, NJ, USA).

Techniques: Labeling

Journal: The FASEB Journal

Article Title: Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1

doi: 10.1096/fj.11-187005

Figure Lengend Snippet: DUB Chip as a tool to identify specific DUBs interacting with KCa3.1. A) BLAP-KCa3.1 was labeled with streptavidin-Alexa488 and incubated for the times indicated at 37°C. B) Cells were lysed in the presence of GST-TUBE2; the lysates were pulled down on GST beads, eluted, and hybridized on a DUB Chip (top panel). An interaction between fluorescently-tagged KCa3.1 and specific DUBs was quantified by measuring the fluorescence intensity (see Materials and Methods). DUB Chip data, expressed as relative fluorescence units (RFU), indicates an interaction between ubiquitylated KCa3.1 and both USP2 and USP8, and a weaker association with AMSH (bottom panel). C) Co-IP confirmed the interaction between KCa3.1 and USP8. Cells were cotransfected with myc-tagged KCa3.1 and either the WT or DN form of the GFP-USP8. USP8 was immunoprecipitated using either an anti-GFP Ab (lanes 1–3 and 5–6) or a nonspecific IgG (lanes 4 and 7) and subsequently IB for KCa3.1 with α-myc Ab. Total cell lysates are shown in lanes 8 and 9.

Article Snippet: Antibodies Polyclonal α-streptavidin Ab was obtained from Genscript Corporation (Piscataway, NJ, USA).

Techniques: Labeling, Incubation, Fluorescence, Co-Immunoprecipitation Assay, Dominant Negative Mutation, Immunoprecipitation

Journal: The FASEB Journal

Article Title: Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1

doi: 10.1096/fj.11-187005

Figure Lengend Snippet: USP8 overexpression alters the ubiquitylation and degradation rate of KCa3.1 A) Cells were doubly transfected with BLAP-KCa3.1 and either GFP vector alone or the WT or DN GFP-USP8. KCa3.1 was labeled at the cell surface with streptavidin-Alexa555, and cells were incubated at 37°C for 8 h. In cells overexpressing either form of USP8, KCa3.1 degradation is slowed compared with control cells, as shown by the strong intracellular signal. Moreover, KCa3.1 clearly colocalizes with DN GFP-USP8 subsequent to endocytosis (bottom panel, arrow in overlay). B) Degradation rate of membrane KCa3.1 was quantified as above. Both WT and DN GFP-USP8 caused a significant delay in KCa3.1 degradation rate (bar graph, n=3). *P < 0.05. C) To correlate the degradation in B with the level of KCa3.1 ubiquitylation, cells were prepared as in B and lysed in the presence of GST-TUBE2 at the indicated times, followed by pulldown on GST beads and IB using α-streptavidin Ab. WT USP8 overexpression decreases ubiquitylation of KCa3.1, while DN USP8 prevents deubiquitylation, as compared with control cells.

Article Snippet: Antibodies Polyclonal α-streptavidin Ab was obtained from Genscript Corporation (Piscataway, NJ, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Labeling, Incubation, Control, Membrane

Journal: Human Gene Therapy

Article Title: Chimeric Receptor mRNA Transfection as a Tool to Generate Antineoplastic Lymphocytes

doi: 10.1089/hum.2008.068

Figure Lengend Snippet: CIR mRNA activity under various conditions. CTLs were obtained after short (1 day) or standard (7 days) ex vivo activation with anti-CD3/CD28 beads and IL-2, and were electroporated with CIR mRNA or without mRNA (mock). Surface CIR expression was visualized with a goat anti-mouse (Fab)2 polyclonal antibody conjugated with biotin and streptavidin PerCP (x axes). (A) Expression of anti-CD19 CIR in CTLs activated for 1 day (panel 1) or for 7 days (panel 2) (Elion et al., 2007); expression of anti-CD19 CIR δ(4–1BB) in CTLs activated for 7 days (panel 3). (B) Cytotoxicity of CTLs under the conditions indicated in (A). Targets, CD19+ autologous B cells, were loaded with 51Cr and analyzed at the indicated E:T ratios.

Article Snippet: Surface CIR expression was visualized with a goat anti-mouse (Fab) 2 polyclonal antibody conjugated with biotin and streptavidin PerCP ( x axes). ( A ) Expression of anti-CD19 CIR in CTLs activated for 1 day (panel 1) or for 7 days (panel 2) (Elion et al. , 2007 ); expression of anti-CD19 CIR δ (4–1BB) in CTLs activated for 7 days (panel 3). ( B ) Cytotoxicity of CTLs under the conditions indicated in ( A ).

Techniques: Activity Assay, Ex Vivo, Activation Assay, Expressing