Marilen Benner

CHAPTER 3 66 provides a strong platform for guiding analysis of altered Tregs in clinical conditions for which no systematic functional Treg characterization has been performed. This study provides strong evidence that multiple types of decidual Tregs, including nTregs and iTregs, play a key role in maintaining maternal-fetal immune tolerance during pregnancy. Moreover, the decidual microenvironment contains cell types, particularly EVTs and decidual macrophages, which have mechanisms to stabilize and expand Treg populations. Of importance here is the data demonstrating that blocking the TCR co-receptor CD3 as well as HLA-C during EVT- CD4 T cell co-cultures inhibited the increase in PD1 HI Tregs, suggesting antigen specificity. Further, characterization of decidual Tregs and Teffs in placental materials obtained after spontaneous preterm birth, preeclampsia, and intrauterine infections will accelerate discovery of therapeutic targets to prevent and cure these severe pregnancy complications, as is underway for treatment of many types of cancer and autoimmune diseases. MATERIALS AND METHODS Discarded human placental and decidual materials (gestational age 6-12 weeks) were obtained from women undergoing elective pregnancy termination at a local reproductive health clinic. Term placental tissues (gestational age > 37 weeks) were obtained from healthy women after uncomplicated pregnancy at term delivered by elective cesarean section or uncomplicated spontaneous vaginal delivery at Tufts Medical Center. All tissues were visually inspected for signs of excessive inflammation (including discoloration, large infarctions and foul odor) and only healthy tissues were used for further processing. Peripheral blood leukocytes were isolated from discarded leukopacks from healthy volunteer blood donors at the Massachusetts General Hospital in Boston, MA. All human tissue used for this research was de-identified, discarded clinical material. No clinical information including the fetal sex and sex of blood donors was available for analysis. The Committee on the Use of Human Subjects (the Harvard IRB) determined that this use of placental and decidual material is Not Human Subjects Research. Term placental tissue was collected under a protocol approved by Tufts Health Sciences IRB. All procedures to process these human blood and tissues materials are described in method details. Isolation of T cells and decidual macrophages The procedures to isolate lymphocytes and EVT have recently been described (6) and are also described in detail hereafter. To isolate 1st trimester decidual lymphocytes, villous and decidual tissues from elective pregnancy terminations were macroscopically identified and separated. Decidua parietalis from term pregnancy was collected by removing the amnion and delicately