Marilen Benner

CHAPTER 3 70 CD4 + pT were added directly to the EVT and decidual macrophage cultures or were added on top of the membrane after insertion of a 0.4 mM trans well membrane (Costar). CD4 + pT were harvested after 60 hours and analyzed by Flow cytometry for CD45, CD4, CD25, PD1, TIGIT and intracellular FOXP3 and HELIOS. HCMV Infection of EVT High titer virus stocks of HCMV-AD169-GFP (IE-1-GFP) (a gift from Prof. Donald Coen at Harvard Medical School) was obtained by infecting Human Foreskin Fibroblasts (HFF) (ATCC) and collecting supernatants after 7 days. Supernatants were aliquoted and snap frozen in liquid nitrogen until use. Primary EVT were infected with HCMV at an MOI of 2-4 for 12 hours (41). Infected cells were imaged in a Nikon EclipseTi fluorescence microscope at 20x magnification. HCMV infection of EVT reached > 80% at day 2, and no cytopathic effects on EVT were visible after HCMV (41). For co-culture assays, HCMV infected cells were washed twice after 12 hours of infection and 100.000 CD4 + pT were added. CD4 + pT were harvested after 48 hours and analyzed by Flow cytometry for CD45, CD4, CD25, PD1, TIGIT and intracellular FOXP3. Quantification and statistical analysis All data was analyzed using GraphPad Prism version 6.07 software. To determine differences between 2 paired groups a non-parametric Wilcoxon Signed Rank test was performed (Figure S1E). To determine differences among more than 2 unpaired groups, a nonparametric Kruskal- Wallis test with Dunn’s multiple comparison post-test was performed (Figure 1, 2, 3, and 5; Figure S3, S4, and S7). For the Dunn’s post-test the mean ranks were compared to control column A (Figure 2B 2G and 3; Figure S4 and S7), column B (Figure 5D-5F) or with the mean rank of every column (Figure 1, 5A-5C, and 5G; Figure S3). P values < 0.05 were considered to denote significant differences. *p < 0.05; **p < 0.01; *** p < 0.005 are indicated within each figure. Sample size indicates biological replicates of individual placental of blood cell isolates and are indicated indicted in each figure legend. Sample sizes were not determined beforehand. ACKNOWLEDGEMENTS We thank Joyce Lavecchio and Silvia Ionescu for help with cell sorting; Donald Coen, Harvard Medical School, Boston, MA, for providing HCMV-AD169-GFP; Ada Taymoori and the research team at Tufts Medical Center for all efforts col lecting placental materials; and Frans Claas, Daniela Cipolletta, and all past and current lab members for their helpful discussions. This work was supported by Strominger Lab departmental funds and the March of Dimes grant 6-FY14- 453. M.B. was supported by the Radboud University Honors Academy, Nora Baart Stichting, and a Radboud Institute for Molecular Life Sciences grant.