Journal: PLoS Biology

Article Title: Binding of Superantigen Toxins into the CD28 Homodimer Interface Is Essential for Induction of Cytokine Genes That Mediate Lethal Shock

doi: 10.1371/journal.pbio.1001149

Figure Lengend Snippet: (A, B) p 12B (10 µg/ml) inhibits SEB-mediated induction in human PBMC of IL2 , IFN-γ , and TNF-α mRNA (autoradiograms) and of protein (graphs; data are shown as means ± SEM ( n = 3 experiments)) (A) but not of IL4 and IL10 (B). mRNA was quantitated by RNase protection analysis; β-actin mRNA indicates equal loading of RNA. (C) In SEB structure (pdb3seb.ent), amino acid residues in the 150–161 β-strand(8)/hinge/α-helix(4) domain are shown in purple, with solvent-accessible residues N151, K153, and K154 in orange. Residues contacting MHC-II are colored red; those contacting the TCR are colored green . (D) p 12B inhibits induction of IL2 and IFN-γ mRNA in PBMC by αCD28 mAb. PBMC were induced by αCD28 (2.5 µg/ml) alone or with 10 µg/ml p 12B . To show that αCD28 does not bind p 12B , p 12B and CD28-Fc were immobilized and binding of mouse αCD28 was assayed. (E) p 12B fails to inhibit induction of IL2 , IFN-γ , and TNF-α genes by αCD3. PBMC were incubated with αCD3 (0.1 µg/ml) alone or with 10 µg/ml p 12B . mRNA and secreted cytokines were quantitated. (F) p 12B inhibits αCD28/αCD3-mediated induction of IL2 , IFN-γ , and TNF-α genes but not of IL10. PBMC were incubated with αCD3, αCD28, or both, with or without 10 µg/ml p 12B . mRNA and secreted cytokines were determined.

Article Snippet: Mouse γ 1 anti-SEB mAb (MB2B33, Toxin Technology), horseradish peroxidase-conjugated goat anti-mouse IgG (KPL); mouse IgG1 anti-human CD28 mAb (MAB342, clone 37407), mouse IgG1 anti-human CD3 epsilon mAb (clone UCHT1), mouse IgG1 anti-human B7-1/CD80 mAb (clone 37710) and anti-human B7-2/CD86 mAb (clone 37301), goat IgG anti-human CD28 polyclonal Ab (AF-342-PB) and goat IgG anti-human B7-2/CD86 polyclonal Ab (AF-141-NF), from R&D Systems.

Techniques: Solvent, Binding Assay, Incubation

Journal: PLoS Biology

Article Title: Binding of Superantigen Toxins into the CD28 Homodimer Interface Is Essential for Induction of Cytokine Genes That Mediate Lethal Shock

doi: 10.1371/journal.pbio.1001149

Figure Lengend Snippet: (A) Phage display peptides selected by affinity for the SEB binding site in CD28 are SEB antagonists that protect mice from killing by SEB. PBMC were induced with SEB alone or with 0.1 µg/ml p c3 , p e12 , or p c9 , a negative control. IL2 and IFN-γ mRNA are shown; β-actin mRNA indicates equal loading of RNA. For p e12 , IL10 was determined (data are shown as means ± SEM ( n = 3 experiments)). Mice ( n = 10 per group) were challenged with 6 µg SEB alone or with 0.2 µg p e12 or 0.5 µg p c3 ; p for survival, 10 −4 . (B–D) Binding of SEB to cell surface CD28. Representative fields of confocal microscopy are shown. In (B), HEK293-T cells were transfected to express CD28-GFP fusion protein (green) and after 48 h incubated for 1 h with Alexa-Fluor-633-labeled SEB (red). In (C), BHK-21 hamster cells were transfected with CD28 cDNA vector and after 48 h incubated successively for 30 min with labeled SEB (red), goat polyclonal αCD28, and Cy2-labeled donkey anti-goat IgG (green). In (D), BHK-21 cells were transfected to express CD28 and after 48 h incubated first with goat polyclonal αCD28 and Cy2-labeled donkey anti-goat IgG (top) and only then with labeled SEB (bottom). (E) Binding of SEB to CD28. SEB, lysozyme, and polyclonal αCD28 (Ab) were separated on duplicate SDS-PAGE gels. Coomassie blue staining (top); far-western blot with CD28-Fc (bottom); M, size marker.

Article Snippet: Mouse γ 1 anti-SEB mAb (MB2B33, Toxin Technology), horseradish peroxidase-conjugated goat anti-mouse IgG (KPL); mouse IgG1 anti-human CD28 mAb (MAB342, clone 37407), mouse IgG1 anti-human CD3 epsilon mAb (clone UCHT1), mouse IgG1 anti-human B7-1/CD80 mAb (clone 37710) and anti-human B7-2/CD86 mAb (clone 37301), goat IgG anti-human CD28 polyclonal Ab (AF-342-PB) and goat IgG anti-human B7-2/CD86 polyclonal Ab (AF-141-NF), from R&D Systems.

Techniques: Binding Assay, Negative Control, Confocal Microscopy, Transfection, Incubation, Labeling, Plasmid Preparation, SDS Page, Staining, Far Western Blot, Marker

Journal: PLoS Biology

Article Title: Binding of Superantigen Toxins into the CD28 Homodimer Interface Is Essential for Induction of Cytokine Genes That Mediate Lethal Shock

doi: 10.1371/journal.pbio.1001149

Figure Lengend Snippet: (A and B) Binding of CD28-Fc in 2-fold increments from 0.25 µM to immobilized SEB (A) and from 0.125 µM to p 12C (B). (C) Binding of CD28-Fc (2 µM) to p 12C without SEB (top curve) or with SEB in 2-fold increments from 0.78 µM (6 lower curves, top to bottom). (D) Binding of 2 µM PD1-Fc and CD28-Fc to immobilized SEB. (E) Binding of CD28-Fc in 2-fold increments from 0.125 µM to scrambled p 12Csc (EKAKYTQLVKDN). (F–I) Mutations in SEB β-strand(8)/hinge/α-helix(4) domain peptides reduce binding of 2 µM CD28-Fc. SPR responses are compared for immobilized p 12C and p SEB (TNKKKVTAQELD), p SEBK (TN A KKVTAQELD) (F), p 12K (YN A KKATVQELD) (G), p 12YK ( A N A KKATVQELD) (H), and p 12KD (YN A KKATVQEL A ) (I). Representative SPR responses are shown.

Article Snippet: Mouse γ 1 anti-SEB mAb (MB2B33, Toxin Technology), horseradish peroxidase-conjugated goat anti-mouse IgG (KPL); mouse IgG1 anti-human CD28 mAb (MAB342, clone 37407), mouse IgG1 anti-human CD3 epsilon mAb (clone UCHT1), mouse IgG1 anti-human B7-1/CD80 mAb (clone 37710) and anti-human B7-2/CD86 mAb (clone 37301), goat IgG anti-human CD28 polyclonal Ab (AF-342-PB) and goat IgG anti-human B7-2/CD86 polyclonal Ab (AF-141-NF), from R&D Systems.

Techniques: Binding Assay

Journal: PLoS Biology

Article Title: Binding of Superantigen Toxins into the CD28 Homodimer Interface Is Essential for Induction of Cytokine Genes That Mediate Lethal Shock

doi: 10.1371/journal.pbio.1001149

Figure Lengend Snippet: (A) CTLA-4/B7-2 complex (1I85.pdb; ) and the dimer interface in CTLA-4. B7-2 is shown in purple and CTLA-4 in blue, with MYPPPY in yellow, YVIDPE (HVKGKH in CD28) in red, and VVLASS (MLVAYD in CD28) in green, as in the sequence alignment of the extracellular domains of human CD28 and CTLA-4. Amino acids at the crystallographic dimer interface of CD28 are shown in blue. Positions of peptides are underlined. (B) p 1TA antagonizes induction of IFN-γ mRNA by αCD28. PBMC were induced by αCD28 (2.5 µg/ml) alone or with 10 µg/ml of CD28-Fc or p 1TA . IFN-γ and β-actin mRNA was determined. (C–E) CD28 dimer interface peptides p 1TA and p 2TA (0.1 µg/ml) inhibit induction by SEB of IL2 and IFN-γ mRNA (C) and IL2, IFN-γ, and TNF-α (D and E) in PBMC (data are shown as means ± SEM ( n = 3 experiments)). (F and G) Induction of IL2 and IFN-γ mRNA by SEA (F) and TSST-1 (G) alone or with 0.1 (F) or 10 µg/ml peptide (G); β-actin mRNA indicates equal loading of RNA.

Article Snippet: Mouse γ 1 anti-SEB mAb (MB2B33, Toxin Technology), horseradish peroxidase-conjugated goat anti-mouse IgG (KPL); mouse IgG1 anti-human CD28 mAb (MAB342, clone 37407), mouse IgG1 anti-human CD3 epsilon mAb (clone UCHT1), mouse IgG1 anti-human B7-1/CD80 mAb (clone 37710) and anti-human B7-2/CD86 mAb (clone 37301), goat IgG anti-human CD28 polyclonal Ab (AF-342-PB) and goat IgG anti-human B7-2/CD86 polyclonal Ab (AF-141-NF), from R&D Systems.

Techniques: Sequencing

Journal: PLoS Biology

Article Title: Binding of Superantigen Toxins into the CD28 Homodimer Interface Is Essential for Induction of Cytokine Genes That Mediate Lethal Shock

doi: 10.1371/journal.pbio.1001149

Figure Lengend Snippet: (A) Mice ( n = 10 per group) were challenged with 6 µg SEB alone or with 1 µg p 1TA , 5 µg p 12B , or 1 µg scrambled peptide p 1TAsc (CHGHLVPKK), or with 0.2 µg p 2TA or p 2TAsc (ASMDYPVL); controls without SEB received 25 µg peptide; p for survival, 2×10 −8 (p 1TA ), 0.38 (p 1TAsc ), 3×10 −6 (p 2TA ), and 0.19 (p 2TAsc ). PBMC were induced with SEB alone or with 1 µg/ml p 1TA or p 1TAsc ; IFN-γ and β-actin mRNA was determined. (B–G) Representative SPR responses for binding of SEB to immobilized CD28-Fc, p 1TA , and p 2TA (B–D), of SEB to p 2TAsc or p2TA (E) and of SEA to p 1TA and p 2TA (F and G). Graphical fits to the binding curves are presented in red color; kinetic parameters show the specificity of these interactions . Analyte concentrations increased in 2-fold increments from 0.156 (C and F), 0.312 (G), 1.56 (B), and 3.12 µM (D); in (E), binding of p 2TA and p 2TAsc is shown for the highest of 5 concentrations tested, 10 µM. In (B), curves in blue color show binding of ribonuclease A in four 2-fold increments from 3.12 µM to immobilized CD28-Fc. (H) Peptide mimetics of the CD28 dimer interface and of the β-strand(8)/hinge/α-helix(4) domain in SEB inhibit binding of SEB to cell-surface CD28. 293-T cells were transfected to express CD28 and after 36 h incubated for 1 h without addition (CD28) or with 0.1 µg/ml αCD28, anti-B7-2 or 10 µg/ml p 1TA , p 2TA , p 12A , or its scrambled form p 12Asc (EKAKYTQLVKDN), before further incubation for 1 h with 15 µg/ml wt SEB. Cells were washed 3 times with cold phosphate-buffered saline before lysis. Western blot of equal amounts of total cell protein (Bradford assay) with αSEB mAb and horseradish peroxidase-conjugated goat anti-mouse IgG shown is from a representative experiment. Bar graph shows intensity of bands for three independent experiments ± SD. U, untransfected; EV, empty vector. (I) IFN-γ, TNF-α, and IL10 (data are shown as means ± SEM ( n = 3)) in medium from PBMC induced with SEB alone or with 0.1 µg/ml p 3TA , p 4TA , or p 5TA .

Article Snippet: Mouse γ 1 anti-SEB mAb (MB2B33, Toxin Technology), horseradish peroxidase-conjugated goat anti-mouse IgG (KPL); mouse IgG1 anti-human CD28 mAb (MAB342, clone 37407), mouse IgG1 anti-human CD3 epsilon mAb (clone UCHT1), mouse IgG1 anti-human B7-1/CD80 mAb (clone 37710) and anti-human B7-2/CD86 mAb (clone 37301), goat IgG anti-human CD28 polyclonal Ab (AF-342-PB) and goat IgG anti-human B7-2/CD86 polyclonal Ab (AF-141-NF), from R&D Systems.

Techniques: Binding Assay, Transfection, Incubation, Saline, Lysis, Western Blot, Bradford Assay, Plasmid Preparation

Journal: PLoS Biology

Article Title: Binding of Superantigen Toxins into the CD28 Homodimer Interface Is Essential for Induction of Cytokine Genes That Mediate Lethal Shock

doi: 10.1371/journal.pbio.1001149

Figure Lengend Snippet: (A) Induction of cytokine genes by wt and tk2 mutant SEB. PBMC were induced with 1 ng/ml wt or tk2 SEB; mRNA (autoradiograms) and secreted cytokines (graphs; data are shown as means ± SEM ( n = 3)) are depicted. (B) Lack of lethality of tk2 SEB. Mice ( n = 5 per group) were challenged with 10 µg wt or tk2 SEB; p for survival, 10 −5 . (C) Affinity of tk2 SEB for CD28. Representative SPR responses for binding of wt and tk2 SEB to immobilized CD28-Fc, p 1TA , and p 2TA are shown for 5 µM wt and tk2 SEB or RNase A to facilitate comparison; kinetic data were collected for five 2-fold increments in protein concentration from 1.25 µM . (D) Dominant-negative phenotype of tk2 SEB. PBMC were induced with 1 ng/ml wt SEB, 0.1 ng/ml tk2 SEB, or both; secreted cytokines were determined as shown (data are shown as means ± SEM ( n = 3)).

Article Snippet: Mouse γ 1 anti-SEB mAb (MB2B33, Toxin Technology), horseradish peroxidase-conjugated goat anti-mouse IgG (KPL); mouse IgG1 anti-human CD28 mAb (MAB342, clone 37407), mouse IgG1 anti-human CD3 epsilon mAb (clone UCHT1), mouse IgG1 anti-human B7-1/CD80 mAb (clone 37710) and anti-human B7-2/CD86 mAb (clone 37301), goat IgG anti-human CD28 polyclonal Ab (AF-342-PB) and goat IgG anti-human B7-2/CD86 polyclonal Ab (AF-141-NF), from R&D Systems.

Techniques: Mutagenesis, Binding Assay, Comparison, Protein Concentration, Dominant Negative Mutation

Journal: PLoS Biology

Article Title: Binding of Superantigen Toxins into the CD28 Homodimer Interface Is Essential for Induction of Cytokine Genes That Mediate Lethal Shock

doi: 10.1371/journal.pbio.1001149

Figure Lengend Snippet: Structural model for Th1 cell activation by superantigens through simultaneous binding to CD28, MHC-II, and TCR. SEC3 (cyan), a close relative of SEB, in complex with TCR Vβ (green) and MHC-II (red) extracellular domains (1jck.pdb; ). The SEC3 β-strand/hinge/α-helix domain in magenta is freely accessible to the N-terminal 118-residue portion of the CD28 extracellular domain (1yjd.pdb; ). CD28 homodimer interface peptides are shown: p 1TA (HVK resolved in 1yjd.pdb is red), p 2TA (green), p 3TA (pink), p 4TA (grey), and p 5TA (SNGTII is blue); MYPPPY is yellow. CD28 and TCR are oriented such that their trans-membrane domains (not shown) can enter the T cell at top; the MHC-II is oriented towards the antigen-presenting cell at bottom; for simplicity, cell surfaces are not shown.

Article Snippet: Mouse γ 1 anti-SEB mAb (MB2B33, Toxin Technology), horseradish peroxidase-conjugated goat anti-mouse IgG (KPL); mouse IgG1 anti-human CD28 mAb (MAB342, clone 37407), mouse IgG1 anti-human CD3 epsilon mAb (clone UCHT1), mouse IgG1 anti-human B7-1/CD80 mAb (clone 37710) and anti-human B7-2/CD86 mAb (clone 37301), goat IgG anti-human CD28 polyclonal Ab (AF-342-PB) and goat IgG anti-human B7-2/CD86 polyclonal Ab (AF-141-NF), from R&D Systems.

Techniques: Activation Assay, Binding Assay, Residue, Membrane