Fig 2: SEB binds CD28 through its ß-strand(8)/hinge/a-helix(4) domain.(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 p12C (B). (C) Binding of CD28-Fc (2 µM) to p12C 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 p12Csc (EKAKYTQLVKDN). (F–I) Mutations in SEB ß-strand(8)/hinge/a-helix(4) domain peptides reduce binding of 2 µM CD28-Fc. SPR responses are compared for immobilized p12C and pSEB (TNKKKVTAQELD), pSEBK (TNAKKVTAQELD) (F), p12K (YNAKKATVQELD) (G), p12YK (ANAKKATVQELD) (H), and p12KD (YNAKKATVQELA) (I). Representative SPR responses are shown.

Fig 3: Peptide mimetics of the CD28 dimer interface are superantigen antagonists.(A) CTLA-4/B7-2 complex (1I85.pdb; [1]) 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 [21] are shown in blue. Positions of peptides are underlined. (B) p1TA antagonizes induction of IFN-? mRNA by aCD28. PBMC were induced by aCD28 (2.5 µg/ml) alone or with 10 µg/ml of CD28-Fc or p1TA. IFN-? and ß-actin mRNA was determined. (C–E) CD28 dimer interface peptides p1TA and p2TA (0.1 µg/ml) inhibit induction by SEB of IL2 and IFN-? mRNA (C) and IL2, IFN-?, and TNF-a (D and E) in PBMC (data are shown as means ± SEM (n = 3 experiments)). (F and G) Induction of IL2 and IFN-? mRNA by SEA (F) and TSST-1 (G) alone or with 0.1 (F) or 10 µg/ml peptide (G); ß-actin mRNA indicates equal loading of RNA.

Fig 4: SEB binds to CD28.(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 pc3, pe12, or pc9, a negative control. IL2 and IFN-? mRNA are shown; ß-actin mRNA indicates equal loading of RNA. For pe12, 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 pe12 or 0.5 µg pc3; 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 aCD28, 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 aCD28 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 aCD28 (Ab) were separated on duplicate SDS-PAGE gels. Coomassie blue staining (top); far-western blot with CD28-Fc (bottom); M, size marker.

Fig 5: Superantigen mimetic peptide blocks SEB action and CD28 signaling.(A, B) p12B (10 µg/ml) inhibits SEB-mediated induction in human PBMC of IL2, IFN-?, and TNF-a 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; ß-actin mRNA indicates equal loading of RNA. (C) In SEB structure (pdb3seb.ent), amino acid residues in the 150–161 ß-strand(8)/hinge/a-helix(4) domain are shown in purple, with solvent-accessible residues [36] N151, K153, and K154 in orange. Residues contacting MHC-II are colored red; those contacting the TCR are colored green [37]. (D) p12B inhibits induction of IL2 and IFN-? mRNA in PBMC by aCD28 mAb. PBMC were induced by aCD28 (2.5 µg/ml) alone or with 10 µg/ml p12B. To show that aCD28 does not bind p12B, p12B and CD28-Fc were immobilized and binding of mouse aCD28 was assayed. (E) p12B fails to inhibit induction of IL2, IFN-?, and TNF-a genes by aCD3. PBMC were incubated with aCD3 (0.1 µg/ml) alone or with 10 µg/ml p12B. mRNA and secreted cytokines were quantitated. (F) p12B inhibits aCD28/aCD3-mediated induction of IL2, IFN-?, and TNF-a genes but not of IL10. PBMC were incubated with aCD3, aCD28, or both, with or without 10 µg/ml p12B. mRNA and secreted cytokines were determined.