Marilen Benner

CHAPTER 3 56 These three CD4 + T cell types were purified by FACS sort and analyzed for their ability to produce the pro- and anti-inflammatory cytokines IFN ɣ , IL-2, and IL-10 upon phorbol 12-myristate 13-acetate (PMA)/Ionomycin stimulation. PD1 HI T cells from all tissues expressed the highest levels of IL-10 and coexpressed IFNg (Figure S3), suggesting a resemblance to Tr1 (30). CD25 HI FOXP3 + cells of all tissue compartments expressed the lowest levels of IL-10, IFN ɣ , and IL-2, whereas TIGIT + cells expressed high levels of IFN ɣ and IL-2 and low levels of IL-10. A limitation of this study is the lack of clinical information on the blood and tissues used for experiments that may have impacts on Treg phenotypes and contribute to the observed variation in Treg proportions. However, a previous report did not find an influence of clinical variables such as mode of delivery and fetal sex on the presence of CD4 + CD25 dim - activated T cells and CD4+CD25 HI FOXP3+ Tregs in decidual tissues of term pregnancy (3). CD25 HI FOXP3+, PD1 HI , and TIGIT+ suppress proliferation of CD4+ and CD8+ Teff cells To determine the capacity of CD25 HI FOXP3, PD1 HI , and TIGIT + T cells to suppress proliferation of effector T cells (Teffs), carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4 + or CD8 + Teffs were stimulated with anti-CD3 and -CD28 beads in the presence or absence of sample- matched Tregs for 4 days in a 1:2 Treg to Teff ratio. CFSE profiles were analyzed to determine the percentage of undivided cell (generation 0) and the average number of divisions per cell (division index) within the CD4 + and CD8 + Teffs cultured with and without Tregs (Figure 2A). All three CD4 + Treg types in decidual samples of 6–12 weeks suppressed proliferation of CD4 + Teffs. This is illustrated by the significant increase in the percentage of undivided cells (Figure 2B), and a significant decrease of the division index (Figure 2E) upon co-culture of Tregs and Teffs, compared to stimulated Teff cultured alone. Additionally, purified PD1 dim cells from 6-12 week decidual tissues did not suppress proliferation of CD4 + T cells (Figure S4A). CD25 HI FOXP3 + T cells also suppressed proliferation of sample-matched CD8 + Teffs, whereas PD1 HI and TIGIT + cells did not consistently reduce CD8 + Teff proliferation (Figures 2C and 2F). Analysis of the three T cell populations from term placenta d.parietalis demonstrated that these cells had a reduced capacity to suppress CD4 + Teff proliferation, compared to their first-trimester counterparts (Figures 2D and 2G), whereas peripheral blood CD25 HI and PD1 HI , but not TIGIT+ cells, suppressed proliferation of CD8 + Teffs (Figures S4B-S4D). To determine if IL-10 secretion by PD1 HI Tregs is their predominant mechanism to suppress proliferation of CD4 + Teffs, as was previously shown for Tr1 cells (30, 31), anti-IL-10R blocking antibodies or immunoglobulin G (IgG) control antibodies were added to anti-CD3 and -CD28 stimulated CFSE- labeled CD4 + Teffs cultured alone or with sample-matched CD25 HI or PD1 HI cells. Indeed, addition of IL-10R antibodies abrogated the suppressive capacity of PD1 HI cells but not of the CD25 HI cells that were added in parallel control cultures (Figure 2H).