Journal: Cell reports

Article Title: Antigen-Presenting Cell-Intrinsic PD-1 Neutralizes PD-L1 in cis to Attenuate PD-1 Signaling in T Cells

doi: 10.1016/j.celrep.2018.06.054

Figure Lengend Snippet: (A) Left: cartoon showing a cell culture assay in which PD-1 transduced Jurkat cells were stimulated with three types of antigen-loaded Raji B cells: (1) parental Raji cells that express neither PD-L1 nor PD-1, (2) Raji cells transduced with only PD-L1 (PD-L1+), and (3) Raji transduced with both PD-L1 and PD-1 (PD-L1+/PD-1+). On the right are FACS histograms showing PD-1 and PD-L1 surface expression of parental Jurkat cells, PD-1–mGFP transduced Jurkat (PD-1+) cells, parental Raji cells, PD-L1–mCherry transduced Raji (PD-L1+) cells, and Raji cells co-transduced with both PD-1–mGFP and PD-L1–mCherry (PD-L1+/PD-1+). a.u., arbitrary units. (B–D) Jurkat cells expressing PD-1–mGFP (PD-1+) were conjugated with Raji B cells (parental) (B), Raji cells transduced with only PD-L1–mCherry (PD-L1+) (C), or Raji cells co-transduced with PD-L1–mCherry and unlabeled PD-1 (PD-L1+/PD-1+) (D), as illustrated in the cartoon on the left (CD28 and B7 are omitted in this cartoon for simplicity). Shown on the right are confocal images of the cell conjugate acquired 2 min after cell-cell contact. mGFP and mCherry signals are shown as green and magenta, respectively. Scale bars, 10 µm. (E) Bar graph summarizing the interface enrichment indices (calculated as described in STAR Methods) of the three conditions shown in (B). Data are shown as mean ± SEM; n = 35 cells from three independent experiments. See also . (F) A representative western blot showing the levels ZAP70-Y493 phosphorylation and p85 (PI3K regulatory subunit) co-immunoprecipitated (IP) with CD28 from the lysates of the indicated Jurkat-Raji co-culture. Jurkat cells expressing PD-1–mGFP (PD-1+) were stimulated with Raji cells (parental), Raji transduced with PD-L1–mCherry (PD-L1+), or Raji cells co-transduced with PD-L1–mCherry and PD-1–mGFP (PD-L1+/PD-1+); the times at which the co-culture was lysed are indicated (STAR Methods). WCL, whole cell lysate. (G and H) Bar graphs summarizing immunoblots in (F), including pY493-ZAP70 immunoblot (G) and CD28 coIP p85 immunoblot (H). The optical density corresponding to each band was quantified by ImageJ and normalized to the conditions in which parental Raji cells were used as the APCs. Data are presented as mean ± SEM from three independent replicates. (I) Bar graph summarizing IL-2 levels in the medium of the indicated Jurkat-Raji co-culture, as described in (B)–(D), 24 hr after cell-cell contact. Data are presented as mean ± SEM from three independent measurements, with each run performed in triplicate.

Article Snippet: ZAP70 pY493 antibody , Cell Signaling Technology , Cat # 2704S; RRID: AB_2217457.

Techniques: Cell Culture, Transduction, Expressing, Western Blot, Phospho-proteomics, Immunoprecipitation, Co-Culture Assay

Journal: Cell reports

Article Title: Antigen-Presenting Cell-Intrinsic PD-1 Neutralizes PD-L1 in cis to Attenuate PD-1 Signaling in T Cells

doi: 10.1016/j.celrep.2018.06.054

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ZAP70 pY493 antibody , Cell Signaling Technology , Cat # 2704S; RRID: AB_2217457.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay, Software, Imaging

Journal: American journal of hematology

Article Title: Igbeta(CD79b) mRNA expression in chronic lymphocytic leukaemia cells correlates with immunoglobulin heavy chain gene mutational status but does not serve as an independent predictor of clinical severity.

doi: 10.1002/ajh.20885

Figure Lengend Snippet: Figure 6. Comparison of normalized Igb mRNA levels and ZAP70 expression in CLL patients. As in Fig. 4, the normalized Igb mRNA levels are shown for individual patients depicted as dots. The individuals are grouped by ZAP70 expression status. Horizontal lines represent the mean in each group. The significance of the pair-wise comparison is indicated by the P value below the graph.

Article Snippet: Immunohistochemistry was performed on five micron paraffin block sections on the DakoCytomation Autostainer (DakoCytomation Corporation, Carpinteria, CA) using the following antibodies: CD3 (Rabbit Polyclonal, 1:60, Neomarkers, Fremont, CA), CD5 (Mouse Monoclonal, clone 4C7, 1:25, Neomarkers), CD20 (Mouse Monoclonal, clone L26, 1:150, DakoCytomation), CD23 (Mouse Monoclonal, clone MHM6, Predilute, DakoCytomation), CD79a (Mouse Monoclonal, clone JCB117, 1:25, DakoCytomation) and ZAP70 (Mouse Monoclonal, clone 2F3.2, 1:80, Upstate Cell Signaling Solutions, Lake Placid, NY).

Techniques: Comparison, Expressing

Journal: American journal of hematology

Article Title: Igbeta(CD79b) mRNA expression in chronic lymphocytic leukaemia cells correlates with immunoglobulin heavy chain gene mutational status but does not serve as an independent predictor of clinical severity.

doi: 10.1002/ajh.20885

Figure Lengend Snippet: Figure 7. Comparison of SHM status, Igb mRNA expression, and ZAP70 expression in CLL with levels of clinical sever- ity. A. Igb mRNA levels. Patients were stratified into low, intermediate, or high disease severity groups as described in materials and methods. The bars represent the percentage of patients in each clinical category that had high Igb mRNA levels. (n = 33) B. IgVH gene mutation. As in A. (n = 33) C. ZAP70 expression. As in A. (n = 29).

Article Snippet: Immunohistochemistry was performed on five micron paraffin block sections on the DakoCytomation Autostainer (DakoCytomation Corporation, Carpinteria, CA) using the following antibodies: CD3 (Rabbit Polyclonal, 1:60, Neomarkers, Fremont, CA), CD5 (Mouse Monoclonal, clone 4C7, 1:25, Neomarkers), CD20 (Mouse Monoclonal, clone L26, 1:150, DakoCytomation), CD23 (Mouse Monoclonal, clone MHM6, Predilute, DakoCytomation), CD79a (Mouse Monoclonal, clone JCB117, 1:25, DakoCytomation) and ZAP70 (Mouse Monoclonal, clone 2F3.2, 1:80, Upstate Cell Signaling Solutions, Lake Placid, NY).

Techniques: Comparison, Expressing, Mutagenesis

Journal: Frontiers in Immunology

Article Title: High-CBD Extract (CBD-X) Downregulates Cytokine Storm Systemically and Locally in Inflamed Lungs

doi: 10.3389/fimmu.2022.875546

Figure Lengend Snippet: CBD-X extract modulates TCR- signaling in human derived CD4 + T cells. CD4 + T cells were isolated from healthy donors by negative magnetic selection with the EasySep Direct Human CD4 + T cells Isolation Kit. Isolated CD4 + T cells were activated with 5 µg/ml anti-CD3 and 5 µg/ml anti-CD28 and treated with 1 or 2 µg/ml CBD-X extract or DMSO as a control. After an hour, the cells were collected and lysed with 100 µl 2x SB+βME. (A) Cell lysates were analyzed for the presence of phosphorylated STAT5, Lck and Zap70 by western blot analysis using pY694-STAT5, total STAT5, pY394-Lck, total Lck, pY319-Zap70 and total Zap70 antibodies. (B) Induction of phosphorylation was quantified relative to the specific total protein expression. Error bars represent the standard deviation of the means of three different donors, and they are expressed as average ± standard deviation (SD). SD were calculated in ratio to activated CD4 (treated with Anti-CD3/CD28), and data were analyzed by one-way ANOVA (Fisher’s LSD test with values p < 0.05 considered statistically significant, (*p <0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Mouse anti-phospho STAT5 and rabbit anti STAT5, rabbit anti-phospho Lck and mouse anti Lck, rabbit anti-phospho Zap70 and mouse anti–Zap70 as well as mouse anti β-Actin were obtained from Cell Signaling Technology (MA, USA).

Techniques: Derivative Assay, Isolation, Selection, Control, Western Blot, Phospho-proteomics, Expressing, Standard Deviation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Abnormal T cell receptor signal transduction of CD4 Th cells in X-linked lymphoproliferative syndrome.

doi: 10.4049/jimmunol.167.5.2657

Figure Lengend Snippet: FIGURE 5. Tyrosine phosphorylation of cellular proteins and SAP-as- sociated protein. A, Enhanced tyrosine phosphorylation of CD3 -chain, ZAP70, and Cbl in XLP CD4 T cells. A total of 1 107 HVS-transformed JJ and Huami CD4 T cells were stimulated with an anti-CD3 Ab at 37°C for 1 min and lysed. Precleared cell lysates were used for immunoprecipi- tation with anti-CD3, ZAP70, or Cbl Ab, followed by immunoblot with anti-phosphotyrosine Ab. B, Interaction of SAP with a 75-kDa tyrosine- phosphorylated protein. A total of 3 107 of HVS-transformed JJ or Huami T cells were stimulated with or without an anti-CD3 Ab at 37°C for 1 min. Precleared whole-cell lysates were used for immunoprecipitation with an anti-SAP Ab. Polypeptides of anti-SAP immune complexes were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and reacted with an anti-phosphotyrosine Ab. Arrows indicate the tyrosine- phosphorylated 75-kDa protein. The data were reproduced in three inde- pendent experiments.

Article Snippet: Sources of commercial Abs are as follows: anti-CD3 (6B10.2), anti-associated protein 70 (ZAP70) (LR), anti-Cbl (C-15), anti-SAP (FL-128), anti-SLAM (N-19), anti-JNK1 (C-17), and anti-glycogen synthase kinase (GSK)-3 (0011-A) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); HRP-conjugated anti-phosphotyrosine (4G10) was obtained from Upstate Biotechnology (Lake Placid, NY); and anti-phospho-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182), anti-p38 MAPK, anti-phosphostress-activated protein kinase/JNK (Thr183/Tyr185), anti-phospho-Akt (Ser473), anti-Akt, and anti-phospho-GSK-3 and - (Ser21/9) were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Phospho-proteomics, Transformation Assay, Western Blot, Immunoprecipitation, SDS Page, Membrane