Article Figures & Data
Figures
- FIGURE 1.
Human CD46 and streptococcal M proteins. A, CD46 contains four CCP domains, the alternatively spliced O-glycosylated region designated B and C, and a region of unknown function (U) that is followed by a transmembrane domain, cytoplasmic anchor, and one of two possible cytoplasmic tails. B, The M5 and M22 proteins are dimeric coiled-coils. The binding sites for human plasma proteins and of the B and C repeat regions are indicated. FHL-1, factor H-related protein 1; Fbg, fibrinogen; C4BP, C4b-binding protein. C, SDS-PAGE analysis (nonreducing) of proteins (2 μg/lane) used in this study. The microheterogeneity of the M proteins is characteristic of this family of proteins when produced by E. coli and may represent variability at the COOH terminus (16 ). See text for description of these proteins. A mouse mAb IgG was included as a control.
- FIGURE 2.
Streptococcal M proteins induce a Tr1-like cytokine secretion profile in human peripheral blood CD4+ T lymphocytes. A, Streptococcal M5 protein induces a similar cytokine profile as mAbs to CD46. Microtiter wells were coated with mAbs to CD molecules and with pure M5 protein, as indicated, and purified T cells were seeded. The cell supernatants were analyzed at 72 h for secreted cytokines. Arrows indicate the detection of added recombinant human IL-2. B, M5-induced IL-10 production is IL-2 dependent. T cells were incubated under the indicated activation conditions for 72 h, with or without a neutralizing mAb to IL-2. Results are the mean ± SD of three independent experiments. ∗∗, p < 0.001.
- FIGURE 3.
M protein-induced IL-10 production by CD4+ T cells is dependent on the C repeat region of M5 and CD46. A, Analysis of plate-immobilized M proteins and mutant proteins for ability to induce IL-10 production. B, Latex bead-bound M proteins induce IL-10 production. Latex beads coated with anti-CD46 mAbs and/or streptococcal proteins were added to T cells (15 beads:1 T cell) in wells coated with or without anti-CD3 as indicated. C, IL-10 production induced by whole streptococci. T cells were seeded into microtiter wells coated with anti-CD3 and/or anti-CD46 and heat-inactivated bacteria were added as indicated (20 bacteria:1 T cell). S. pyog. M5, bacteria expressing M5; S. pyog. ΔM5, isogenic M5-negative mutant. D—F, Protocols as indicated in A, B, and C, respectively, except for the addition of the indicated amounts of soluble CD46 (sCD46) to the medium at time 0. All supernatants were analyzed at 72 h. Results are the mean ± SD of three independent experiments. ∗∗∗, p < 0.001; ∗, p < 0.05.
- FIGURE 4.
M protein induces a similar morphological phenotype in CD4+ T cells as mAbs to CD46. The T cells were stimulated for 3 days with or without the addition of 10 μg/ml soluble CD46. Original magnification, ×200. Results shown are representative of three independent experiments.
- FIGURE 5.
Activation of CD46 through purified M5 or M5-expressing bacteria induce functional Tr1-like cells. A, M protein-activated T cells inhibit the proliferation of bystander T cells through IL-10. Upper panel, T cells were activated for 3 days with the indicated immobilized mAbs or streptococcal proteins. Supernatants were transferred to freshly purified CD4+ T cells, the mixture was incubated with stimulating Abs or PBS, and proliferation was measured at day 7. Lower panel, As above but with the addition of 10 μg/ml of a neutralizing mAb to IL-10. B, Bacteria-activated T cells inhibit the activation of bystander T cells. Upper panel, T cells were plated onto microtiter plate wells coated with the indicated mAbs and bacteria were added (20 bacteria:1 T cell). At day 3, supernatants were transferred to freshly purified T cells and the mixtures were incubated under the indicated stimulation conditions. Proliferation was measured at day 7. Lower panel, As above but with the addition of a neutralizing anti-IL-10 mAb. Results are the mean ± SD of three independent experiments. Note that, in contrast to CD3/CD28- and CD3/CD46-activated T cells, CD3/Rib-activated T cells do not produce growth factors; thus, their supernatants do not add to the proliferation of freshly purified CD3/CD28-activated T cells. This is also observed for supernatants derived from cells activated with mAbs to CD3 alone (data not shown). ∗∗∗, p < 0.001; ∗, p < 0.05.
- FIGURE 6.
T cells activated with M protein exhibit perforin/granzyme B-dependent cytotoxicity. A, Activation of CD46 through M5 protein induces granzyme B expression. Purified human T cells were incubated with the indicated immobilized mAbs and/or M5 protein for 36 h and granzyme B expression was determined by FACS. Results shown are representative of three independent experiments. B, M protein-activated T cells kill human monocytic U937 cells. Purified T cells were activated for 60 h under the indicated conditions and used as effector cells in a flow-based killing assay against target U937 cells with or without the addition of a function-neutralizing mAb to CD18. Data are representative of three independent experiments showing similar results. ∗∗∗, p < 0.001.
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