Figure 6
Figure 6. CD40L-NBD T cells suppress proliferation of CFSE-labeled CD4+ target cells and are generated via a noncanonical NF-κB pathway–mediated mechanism in DCs. (A) CD40L-NBD T cells suppress proliferation of CFSE-labeled CD4+ target T cells. Naive CD4+ T cells instructed by NBD-treated CD40L-stimulated DCs (CD40L-NBD T cells) were tested for their suppressive activity on proliferation of anti-CD3/CD28–stimulated CFSE-labeled CD4+ target T cells. CFSE profiles of target cells cocultured with CD40L-NBD T cells (filled histogram), target cells cocultured with CD40L-MUT T cells (black line), or target cells alone (gray line) are shown. Histograms are representative profiles from 1 of 5 independent experiments that yielded similar results. (B) Quantification of target cell proliferation in coculture with CD40L-NBD T cells or CD40L-MUT T cells, instructed by DCs in the presence or absence of 1-methyl-tryptophan (MT). Data are expressed as percent proliferation compared with control (medium + CD40L) and represent mean plus or minus SEM from 5 independent experiments (*P < .001). (C) Suppressive capacity of CD40L-NBD T cells is induced by DCs via a noncanonical NF-κB pathway–mediated mechanism. CD40L-NBD DCs were treated with siRNA for IKKα (siIKKα) or control siRNA (siC), and subsequently the suppressive activity of T cells instructed by these DCs (siIKKα or siC CD40L-NBD T cells, respectively) was tested in the same assay as described in panel A. CFSE profiles of target cells cocultured with siC CD40L-NBD T cells (filled histogram) and target cells cocultured with siIKKα CD40L-NBD T cells (gray line) were compared with CD40L-MUT T cells (black line). Shown are representative profiles from 1 of 3 independent experiments that yielded similar results. (D) Quantification of target cell proliferation in coculture with T cells derived from siRNA-treated CD40L-stimulated NBD/MUT DCs. Data are expressed as percent proliferation compared with control (medium + CD40L) and represent mean plus or minus SEM from 3 independent experiments (*P < .001).

CD40L-NBD T cells suppress proliferation of CFSE-labeled CD4+ target cells and are generated via a noncanonical NF-κB pathway–mediated mechanism in DCs. (A) CD40L-NBD T cells suppress proliferation of CFSE-labeled CD4+ target T cells. Naive CD4+ T cells instructed by NBD-treated CD40L-stimulated DCs (CD40L-NBD T cells) were tested for their suppressive activity on proliferation of anti-CD3/CD28–stimulated CFSE-labeled CD4+ target T cells. CFSE profiles of target cells cocultured with CD40L-NBD T cells (filled histogram), target cells cocultured with CD40L-MUT T cells (black line), or target cells alone (gray line) are shown. Histograms are representative profiles from 1 of 5 independent experiments that yielded similar results. (B) Quantification of target cell proliferation in coculture with CD40L-NBD T cells or CD40L-MUT T cells, instructed by DCs in the presence or absence of 1-methyl-tryptophan (MT). Data are expressed as percent proliferation compared with control (medium + CD40L) and represent mean plus or minus SEM from 5 independent experiments (*P < .001). (C) Suppressive capacity of CD40L-NBD T cells is induced by DCs via a noncanonical NF-κB pathway–mediated mechanism. CD40L-NBD DCs were treated with siRNA for IKKα (siIKKα) or control siRNA (siC), and subsequently the suppressive activity of T cells instructed by these DCs (siIKKα or siC CD40L-NBD T cells, respectively) was tested in the same assay as described in panel A. CFSE profiles of target cells cocultured with siC CD40L-NBD T cells (filled histogram) and target cells cocultured with siIKKα CD40L-NBD T cells (gray line) were compared with CD40L-MUT T cells (black line). Shown are representative profiles from 1 of 3 independent experiments that yielded similar results. (D) Quantification of target cell proliferation in coculture with T cells derived from siRNA-treated CD40L-stimulated NBD/MUT DCs. Data are expressed as percent proliferation compared with control (medium + CD40L) and represent mean plus or minus SEM from 3 independent experiments (*P < .001).

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