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. 2016 May 3;24(6):1126–1134. doi: 10.1038/mt.2016.64

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Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy

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Regulatory T-cells (Treg) expanded after alloanergization in lipopolysaccharide (LPS)-rich conditions are enriched with allosuppressive IFN-γ-secreting cells. (a) Frequencies of IFN-γ⁺ cells expressed as percentage of CD4⁺ Treg at baseline, after alloanergization and after allorestimulation in the absence or presence of LPS. Horizontal lines represent median values. P values are for two-tailed t-test. Results are shown for eight HLA-mismatched stimulator-responder pairs. *P < 0.05, **P < 0.01. (b) Frequencies of IL-17A⁺ cells expressed as percentage of Treg at baseline, after alloanergization and after allorestimulation. Horizontal lines represent median values. P values are for two-tailed t-test. Results are shown for eight HLA-mismatched stimulator- responder pairs. ns, not significant. (c) IFN-γ⁺ Treg following alloanergization and allorestimulation upregulate T-bet. Bar chart summarizes mean (±SD) median fluorescence intensity (MFI) of T-bet CD4⁺FOXP3⁺IFN-γ⁺ Treg at baseline and after alloanergization and allorestimulation. Results for three independent experiments are shown. P values are for two-tailed t-test. **P < 0.01, ns, not significant. (d) IFN-γ⁺ Treg following alloanergization and allorestimulation are predominantly helios^neg. Mean (±SD) frequencies of Helios⁺ cells expressed as a percentage of CD4⁺FOXP3⁺IFN-γ⁺ Treg at baseline and after alloanergization and subsequent allorestimulation. Results are for three independent experiments. P values are for two-tailed t-test. *P < 0.05, ns, not significant. (e) Allosuppressive capacity of IFN-γ⁺ and IFN-γ^neg Treg subpopulations after alloanergization and subsequent allorestimulation of healthy donor peripheral blood mononuclear cells (PBMCs) in the presence or absence of LPS. Mean percentage suppression (±SD) of alloproliferative responses of untreated responder cells are shown by purified Treg from allorestimulated alloanergized PBMCs at a ratio of 1:10 Treg: Responder cells. Results are from four independent HLA-mismatched stimulator-responder pairs. Ns, not significant.