Fig. 4
BMDCs induce functional Tregs. GM-CSF-generated BMDCs were pretreated with media or 20 ng/ml GM-CSF for 2 days and/or stimulated with 30 μg/ml N-α-Syn for 1 day prior to co-culture for 5 days with CD4+ T cells. Non-adherent T cells were harvested, stained for expression of CD4, CD25, and Foxp3, and evaluated by flow cytometric analysis. CD4+ gate was drawn to include > 98% of the populations and quadrants were placed to include 98% of the isotype control. a Contour plots of flow cytometric analyses for expression of Foxp3 and CD25 by CD4+ T cells from 5 day co-cultures of BMDCs and CD4+CD25- T cells. b Quantitation of CD25+Foxp3+ Tregs within CD4+ T cell population from BMDC-CD4+ T cell co-cultures or treated with CD3/CD28 transactivation beads. Mean percentages ± SEM of Tregs were determined for 3 replicate co-cultures and evaluated by one-way ANOVA followed by Newman-Keul’s post-hoc test whereby p ≤ 0.05 compared to astarting CD4+ T cells; bmedia-cultured BMDCs/CD4+ T cells; cGM-CSF-cultured BMDCs/CD4+ T cells; dmedia-cultured, N-α-Syn-stimulated BMDCs/CD4+ T cells; and eGM-CSF cultured, N-α-syn stimulated BMDCs/CD4+ T cells. c Tregs were enriched by magnetic bead selection for CD4+CD25+ T cells either from 5 day media co-cultures of BMDCs/CD4+ T cells or from primary isolates of splenic T cells from naïve mice. Tregs were diluted by serial 2-fold dilutions, added to CFSE-labelled CD4+CD25- cells, and stimulated with CD3/CD28 tansactivator beads for 3 days. Cells were harvested and assessed by flow cytometric analysis for Treg activity to suppress T cell proliferation. Linear regression showed that Treg activity correlated with Treg dose for BMDC-induced Tregs (r2 = 0.7941, F = 80.97, DFn = 1, DFd = 21, p < 0.00001) and for primary splenic Tregs (r2 = 0.6815, F = 47.07, DFn = 1, DFd = 22, p < 0.0001). Additionally, comparison of the two regression analyses show that the lines are significantly different (F = 8.52, DFn = 1, DFd = 43, p = 0.0056)