Marilen Benner

CHAPTER 3 58 Figure 3. Decidual Tregs influence cytokine production by CD4+ and CD8+ Teffs. (A–F) Graphs depict absolute change (D) in percentage of IFN- ɣ + (A and D), TNF- α + (B and E) , and IL-10+ (C and F) decidual CD4+ Teffs (top panels) and decidual CD8+ Teffs (bottom panels) upon addition of sample-matched CD25 HI , PD1 HI , or TIGIT+ T cells, compared to CD4+ Teffs and CD8+ Teffs cultured alone. Bars represent median and interquartile range; n = 4–5; *p < 0.05, **p < 0.01. See also Figure S5. CD25 HI FOXP3+ and PD1 HI Tregs influence cytokine production of CD4+ and CD8+ Teffs Next, the capacity of decidual CD25 HI FOXP3 + , PD1 HI , and TIGIT + Tregs to influence production of pro- and anti-inflammatory cytokines by CD4 + and CD8 + Teffs was investigated. CFSE- labeled CD4 + Teffs and CD45-Alexa700 labeled CD8 + Teffs were cultured with or without sample matched Tregs in a 1:1:1 CD4 Teff: CD8 Teff: Treg ratio and stimulated with anti-CD3 and -CD28. At day three, the cells were restimulated with PMA and Ionomycin for 6h in the presence of GolgiStop for the last 4h. CD4 + Teffs, CD8 + Teffs, and Tregs were identified (Fig ure S5A) and analyzed for the presence of intracellular cytokines IFN ɣ , TNF ɑ , and IL-10 (Figure S5B). Addition of CD25 HI cells significantly decreased the percentage of IFN ɣ + and TNF ɑ + CD4 + and IFN ɣ + CD8 + Teffs (Figures 3A,3B, and 3D). Addition of PD1 HI cells significantly increased the percentage of IL-10 + CD4 + and CD8 + Teffs (Figures 3C and 3F) but also increased the percentage of IFN ɣ + CD8 + Teffs (Figure 3D). TIGIT + cells did not consistently change the production of cytokines by CD4 + or CD8 + Teffs (Figure 3). Analysis of cytokine production by Tregs themselves confirmed the overall