Journal: bioRxiv

Article Title: A murine model with JAK2V617F expression in both hematopoietic cells and vascular endothelial cells recapitulates the key features of human myeloproliferative neoplasm

doi: 10.1101/2021.08.24.457585

Figure Lengend Snippet: Decreased marrow hematopoiesis in the Tie2FF1 mice during aging. ( A ) Colony formation assays in marrow cells isolated from young (n=4 mice in each group) and old (n=6-7 mice in each group) Tie2-cre control and Tie2FF1 mice. ( B ) Representative flow cytometry plots showing gating strategy (left) of marrow Lin - cKit + Sca1 + CD150 + CD48 - HSCs frequency (right) in young (n=7 mice in each group) and old (n=5-6 mice in each group) Tie2-cre control and Tie2FF1 mice. ( C-D ) Cell proliferation of marrow Lin-HSPCs isolated from young (C) and old (D) Tie2-cre control and Tie2FF1 mice. Cells were cultured on SFEM medium containing recombinant mouse SCF (100ng/mL), recombinant mouse IL3 (6ng/mL), and recombinant human IL6 (10ng/mL). Data are from one of two independent experiments (with triplicates in each experiment) that gave similar results. * P <0.05

Article Snippet: For analysis of HSC (Lin - cKit + Sca1 + CD150 + CD48 - ) proliferation, Lin - cells were first enriched using the Lineage Cell Depletion Kit (Miltenyi Biotec) before staining with fluorescent antibodies specific for cell surface HSC markers, followed by fixation and permeabilization using the Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA), DNase digestion (Sigma, St. Louis, MO), and anti-BrdU antibody (Biolegend, San Diego, CA) staining to analyze BrdU incorporation .

Techniques: Isolation, Control, Flow Cytometry, Cell Culture, Recombinant

Journal: bioRxiv

Article Title: A murine model with JAK2V617F expression in both hematopoietic cells and vascular endothelial cells recapitulates the key features of human myeloproliferative neoplasm

doi: 10.1101/2021.08.24.457585

Figure Lengend Snippet: Expanded splenic extramedullary hematopoiesis in the Tie2FF1 mice. ( A ) Colony formation assays in spleen cells isolated from young (n=3 mice in each group) and old (n=5-6 mice in each group) Tie2-cre control and Tie2FF1 mice. ( B ) Spleen Lin - cKit + Sca1 + CD150 + CD48 - HSCs frequency in young (n=3 mice in each group) and old (n=5 mice in each group) Tie2-cre control and Tie2FF1 mice. ( C-D ) Cell proliferation of spleen Lin-HSPCs isolated from young (C) and old (D) Tie2-cre control and Tie2FF1 mice. Cells were cultured in SFEM medium containing recombinant mouse SCF (100ng/mL), recombinant mouse IL3 (6ng/mL), and recombinant human IL6 (10ng/mL). Data are from one of two independent experiments (with triplicates in each experiment) that gave similar results. * P <0.05

Article Snippet: For analysis of HSC (Lin - cKit + Sca1 + CD150 + CD48 - ) proliferation, Lin - cells were first enriched using the Lineage Cell Depletion Kit (Miltenyi Biotec) before staining with fluorescent antibodies specific for cell surface HSC markers, followed by fixation and permeabilization using the Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA), DNase digestion (Sigma, St. Louis, MO), and anti-BrdU antibody (Biolegend, San Diego, CA) staining to analyze BrdU incorporation .

Techniques: Isolation, Control, Cell Culture, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Dependence of T Cell Antigen Recognition on the Dimensions of an Accessory Receptor–Ligand Complex

doi:

Figure Lengend Snippet: Expression of elongated and shortened forms of CD48 in I-E k+ CHO cells. (A) Schematic representation of the various forms of CD48 used in this study. Segments derived from mouse CD48 and segments inserted from human CD2 or mouse CD22 are depicted as heavy and light lines, respectively. The asterisk represents the CD22 mutation, R130A. (B) Western blot of the CD48 constructs expressed on I-E k+ CHO cells. Triton X-100 lysates of 10 5 I-E k+ CHO cells expressing no CD48 (CD48 neg) or the indicated form of CD48 were run under reducing conditions and blotted with the mouse CD48 mAb, OX78. Soluble recombinant mouse CD48 with an oligohistidine tag (sCD48his, 50 ng) was included for comparison. Parallel blots of the same samples with an isotype-matched control mAb (OX11) gave no staining (not shown).

Article Snippet: The following mAbs were used: RM2-1, rat anti–mouse CD2, IgG2a ; OX11, rat anti–rat κ chain, IgG2a ; OX78, rat anti–mouse CD48, IgG2a ; IE-D6, mouse anti–mouse I-E k , IgG2a (Serotec); OX12, mouse anti–rat κ chain, IgG2a ; 145-2C11, hamster anti–mouse-CD3 ; A2B4-2, mouse anti-2B4 TCR-α, IgG2a ; and 30-H12, rat anti–mouse Thy1.2, IgG2b (PharMingen).

Techniques: Expressing, Derivative Assay, Mutagenesis, Western Blot, Construct, Recombinant, Comparison, Control, Staining

Journal: The Journal of Experimental Medicine

Article Title: Dependence of T Cell Antigen Recognition on the Dimensions of an Accessory Receptor–Ligand Complex

doi:

Figure Lengend Snippet: Flow cytometry of APCs and T cells. (A) Comparison of I-E k+ CHO cell clones expressing wild-type and elongated forms of CD48. In the left and center columns, cells were labeled with the OX78 (CD48) and IE-D6 (anti–I-E k ) mAbs (thick lines), respectively, or isotype-matched mAbs (left, OX11; middle, OX12) as controls (shaded). In this analysis, OX78 binding was 15–20% lower on CD48-CD2 and CD48-CD22 cells compared with CD48 I-E k+ CHO cells. However, no significant difference was measured in three other analyses (not shown). In the right column, cells were labeled with mCD2-Fc–coated (bold line) or hCTLA4-Fc–coated (shaded) fluorescein beads. (B) Comparison of I-E k+ CHO cells expressing wild-type and shortened CD48, as in A. (C) Comparison of CD2 expression on the different 2B4 cell lines used. Labeling by the CD2 (RM2-1) and the isotype-matched control (OX11) mAbs is represented by bold lines and shaded histograms, respectively. All 2B4 cells expressed virtually identical levels of surface TCR (not shown).

Article Snippet: The following mAbs were used: RM2-1, rat anti–mouse CD2, IgG2a ; OX11, rat anti–rat κ chain, IgG2a ; OX78, rat anti–mouse CD48, IgG2a ; IE-D6, mouse anti–mouse I-E k , IgG2a (Serotec); OX12, mouse anti–rat κ chain, IgG2a ; 145-2C11, hamster anti–mouse-CD3 ; A2B4-2, mouse anti-2B4 TCR-α, IgG2a ; and 30-H12, rat anti–mouse Thy1.2, IgG2b (PharMingen).

Techniques: Flow Cytometry, Comparison, Clone Assay, Expressing, Labeling, Binding Assay, Control

Journal: The Journal of Experimental Medicine

Article Title: Dependence of T Cell Antigen Recognition on the Dimensions of an Accessory Receptor–Ligand Complex

doi:

Figure Lengend Snippet: The CD2/CD48 interaction enhances T cell antigen recognition. (A) Antigen recognition by 2B4.CD2 cells using as APCs untransfected I-E k+ CHO cells (CD48 neg CHO), I-E k+ CHO cells stably transfected with CD48 (CD48 CHO), or CD48 − revertant cells derived from the latter clone (CD48 CHO revertant). In each well, 5 × 10 4 2B4.CD2 cells were mixed with the same number of APCs and the indicated concentrations of peptide. After 18 h of culture, supernatants were analyzed for IL-2. Error bars represent the SE of triplicate cultures. (B) Comparison of antigen recognition by 2B4 cells expressing high levels (2B4.CD2) or very low levels (2B4) of CD2 using CD48 + or CD48 − I-E k+ CHO cells as APCs. Experiment performed as in A. The results were normalized to aid comparison between the different 2B4 cells, letting 100% equal maximal peptide-stimulated IL-2 secretion in the presence of CD48 neg CHOs. (C) Comparison of antigen recognition by 2B4 cells expressing full-length (2B2.CD2) or truncated (2B4.CD2trunc) forms of CD2 cells with CD48 + or CD48 − I-E k+ CHO cells as APCs. Experiment performed as in A. (D) Binding of CD48-coated beads to 2B4.CD2 cells is blocked by a CD2 mAb. 2B4.CD2 T cells were incubated with a mouse CD2 (RM2-1) or control (OX11) mAb before incubation with CD5- (control) or CD48-coated fluorescent beads, followed by flow cytometry. Identical results were obtained with 2B4.CD2trunc T cells.

Article Snippet: The following mAbs were used: RM2-1, rat anti–mouse CD2, IgG2a ; OX11, rat anti–rat κ chain, IgG2a ; OX78, rat anti–mouse CD48, IgG2a ; IE-D6, mouse anti–mouse I-E k , IgG2a (Serotec); OX12, mouse anti–rat κ chain, IgG2a ; 145-2C11, hamster anti–mouse-CD3 ; A2B4-2, mouse anti-2B4 TCR-α, IgG2a ; and 30-H12, rat anti–mouse Thy1.2, IgG2b (PharMingen).

Techniques: Stable Transfection, Transfection, Derivative Assay, Comparison, Expressing, Binding Assay, Incubation, Control, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Dependence of T Cell Antigen Recognition on the Dimensions of an Accessory Receptor–Ligand Complex

doi:

Figure Lengend Snippet: Elongated forms of CD48 inhibit T cell antigen recognition. (A) Antigen recognition by 2B4.CD2 cells using as APCs I-E k+ CHO cells expressing no CD48 (CD48 neg CHO), wild-type CD48 (CD48 CHO), CD48-CD2, or CD48-CD22. The assay was performed as described in the legend to A. The error bars for cultures containing CD48-CD2 and CD48-CD22 I-E k+ CHO cells are smaller than the triangular symbols and cannot be seen. (B) CD48-CD2 or CD48-CD22 inhibit antigen recognition by 2B4 cells expressing full-length (2B2.CD2) or truncated (2B4.CD2trunc) CD2. Assay performed as in A with 10 μM peptide. (C) The stimulatory effects of CD48 and inhibitory effects of CD48-CD2 and CD48-CD22 are reversed by a blocking CD48 mAb. Assay performed as in A with 1 μM peptide except that before the assay, the I-E k+ CHO cells were incubated for 1 h with 10 μg/ml of the CD48 mAb OX78 and the mAb included throughout the assay. The background response (in the absence of peptide) was subtracted from all values. In B and C, the results were normalized to aid comparison between the different 2B4 cells, letting 100% equal maximal peptide-stimulated IL-2 secretion in the presence of CD48 neg CHOs. (D) Revertant (CD48 − ) cells derived from the CD48-CD2 and CD48-CD22 I-E k+ CHO clones do not inhibit the antigen response of 2B4.CD2trunc cells. Revertant cells expressed the same levels of I-E k as their parent clones (not shown). Assay performed as in A.

Article Snippet: The following mAbs were used: RM2-1, rat anti–mouse CD2, IgG2a ; OX11, rat anti–rat κ chain, IgG2a ; OX78, rat anti–mouse CD48, IgG2a ; IE-D6, mouse anti–mouse I-E k , IgG2a (Serotec); OX12, mouse anti–rat κ chain, IgG2a ; 145-2C11, hamster anti–mouse-CD3 ; A2B4-2, mouse anti-2B4 TCR-α, IgG2a ; and 30-H12, rat anti–mouse Thy1.2, IgG2b (PharMingen).

Techniques: Expressing, Blocking Assay, Incubation, Comparison, Derivative Assay, Clone Assay

Journal: The Journal of Experimental Medicine

Article Title: Dependence of T Cell Antigen Recognition on the Dimensions of an Accessory Receptor–Ligand Complex

doi:

Figure Lengend Snippet: Elongated CD48 molecules do not inhibit the response of T cells to antigen-independent stimuli. (A) CHO cells with elongated CD48 do not inhibit T cell activation by PMA/ionomycin. Assay performed as in the legend to A except that, instead of peptide antigen, PMA (50 ng/ml) plus ionomycin (500 ng/ml) was used to stimulate 2B4.CD2trunc T cells. (B) CHO cells with elongated CD48 do not inhibit T cell activation by CD3 mAb. Assay performed as in the legend to A except that, instead of peptide antigen, CD3 mAb 145-2C11 was immobilized to microtiter plates (see Materials and Methods) before addition of 2B4.CD2trunc T cells and APCs. Similar results were obtained when soluble 145-2C11 was used to stimulate the T cells (not shown).

Article Snippet: The following mAbs were used: RM2-1, rat anti–mouse CD2, IgG2a ; OX11, rat anti–rat κ chain, IgG2a ; OX78, rat anti–mouse CD48, IgG2a ; IE-D6, mouse anti–mouse I-E k , IgG2a (Serotec); OX12, mouse anti–rat κ chain, IgG2a ; 145-2C11, hamster anti–mouse-CD3 ; A2B4-2, mouse anti-2B4 TCR-α, IgG2a ; and 30-H12, rat anti–mouse Thy1.2, IgG2b (PharMingen).

Techniques: Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: Dependence of T Cell Antigen Recognition on the Dimensions of an Accessory Receptor–Ligand Complex

doi:

Figure Lengend Snippet: A shortened form of CD48 is able to enhance T cell antigen recognition. Assay performed as in the legend to A using as responders 2B4 cells expressing either full-length (2B4.CD2) or truncated (2B4.CD2trunc) forms of CD2, and as APCs I-E k+ CHO cells expressing no CD48 (CD48 neg CHO), wild-type CD48, or shortened CD48 (CD48d1 CHO).

Article Snippet: The following mAbs were used: RM2-1, rat anti–mouse CD2, IgG2a ; OX11, rat anti–rat κ chain, IgG2a ; OX78, rat anti–mouse CD48, IgG2a ; IE-D6, mouse anti–mouse I-E k , IgG2a (Serotec); OX12, mouse anti–rat κ chain, IgG2a ; 145-2C11, hamster anti–mouse-CD3 ; A2B4-2, mouse anti-2B4 TCR-α, IgG2a ; and 30-H12, rat anti–mouse Thy1.2, IgG2b (PharMingen).

Techniques: Expressing

Journal: The Journal of Experimental Medicine

Article Title: Dependence of T Cell Antigen Recognition on the Dimensions of an Accessory Receptor–Ligand Complex

doi:

Figure Lengend Snippet: Elongated CD48 inhibits TCR downmodulation. TCR downmodulation was measured by flow cytometry on 2B4.CD2 cells after coculture with the indicated I-E k+ CHO cell APCs loaded with various concentrations of MCC peptide. Downmodulation is expressed as the percent reduction in mean TCR levels, with 0% downmodulation equal to the TCR level measured in the absence of peptide. The values shown are the mean ± SD of triplicate cultures. Data shown are representative of three independent experiments.

Article Snippet: The following mAbs were used: RM2-1, rat anti–mouse CD2, IgG2a ; OX11, rat anti–rat κ chain, IgG2a ; OX78, rat anti–mouse CD48, IgG2a ; IE-D6, mouse anti–mouse I-E k , IgG2a (Serotec); OX12, mouse anti–rat κ chain, IgG2a ; 145-2C11, hamster anti–mouse-CD3 ; A2B4-2, mouse anti-2B4 TCR-α, IgG2a ; and 30-H12, rat anti–mouse Thy1.2, IgG2b (PharMingen).

Techniques: Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Dependence of T Cell Antigen Recognition on the Dimensions of an Accessory Receptor–Ligand Complex

doi:

Figure Lengend Snippet: A model explaining the contrasting effects of wild-type and elongated CD48 on T cell antigen recognition. CD2 molecules on T cells and CD48 molecules on I-E k+ CHO APCs interact to form contacts in which the intermembrane separation distance is determined by the dimensions of the CD2/CD48 complex. Wild-type CD48 (left) enhances T cell antigen recognition because the separation distance (∼15 nm) is optimal for TCR engagement of peptide–MHC. Elongated CD48 (CD48-CD22, right) inhibits T cell antigen recognition by forming contacts in which the intermembrane distance (>20 nm) is too great for TCR to engage peptide–MHC.

Article Snippet: The following mAbs were used: RM2-1, rat anti–mouse CD2, IgG2a ; OX11, rat anti–rat κ chain, IgG2a ; OX78, rat anti–mouse CD48, IgG2a ; IE-D6, mouse anti–mouse I-E k , IgG2a (Serotec); OX12, mouse anti–rat κ chain, IgG2a ; 145-2C11, hamster anti–mouse-CD3 ; A2B4-2, mouse anti-2B4 TCR-α, IgG2a ; and 30-H12, rat anti–mouse Thy1.2, IgG2b (PharMingen).

Techniques:

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD48 , REA426 , 50 , 130-106-516 , PE , Miltenyi Biotec.

Techniques: Imaging