Tatiana Takiishi, Hannelie Korf, Tom L. Van Belle, Sofie Robert, Fabio A. Grieco, Silvia Caluwaerts, Letizia Galleri, Isabella Spagnuolo, Lothar Steidler, Karolien Van Huynegem, Pieter Demetter, Clive Wasserfall, Mark A. Atkinson, Francesco Dotta, Pieter Rottiers, Conny Gysemans, Chantal Mathieu
(A) CD25+Foxp3+ cells in PLNs, shown as mean ± sem within the CD4 gate. (B) Histogram overlays of FR4 (top) and CTLA4 (bottom) by CD4+CD25+Foxp3+ cells isolated from PLNs (end of treatment). Inset values: MFI of FR4 expression and percentage of CTLA4+ cells in CD4+CD25+Foxp3+ gate, respectively. Gray histograms: staining on indicated sample; solid lines: isotype staining on nondiabetic control sample. (C–E) In vitro polyclonal suppressor assay. CD4+CD25– Tresps isolated from normoglycemic NOD mice were dye labeled and stimulated using 0.5 μg/ml soluble anti-CD3 and accessory cells for 72 hours in the presence of CD4+CD25+ Tregs isolated from cured mice at the end of the indicated treatment. Results of assays for in vitro suppressive capacity are shown. (C) Proliferation of Tresps, shown as percentage of Tresps that had undergone 2 or more divisions, normalized to Tresp-only culture. (D) Activation of Tresps, shown as MFI of CD44 expression, normalized to Tresp-only culture. (E) ELISA for IL-10. (F) Assay for in vivo suppressive capacity. CD25-depleted splenocytes from diabetic NOD mice were transferred into NOD/SCID mice without (white circles) or with CD4+CD25+ cells from CT-cured (white squares) or CT-non-cured (black squares) mice. Shown is the diabetes incidence in recipients. Statistical calculation using Mann-Whitney test (#P < 0.05, ##P < 0.01 versus week 6 CT; †P < 0.05, ††P < 0.01, †††P < 0.001 versus week 14 CT), t test (C–E), and Mantel-Cox log-rank (F). Results in C–E are representative of at least 3 experiments (each at least 3 treated mice). *P < 0.05, ***P < 0.001.