P2.H159: IDENTIFICATION AND MULTIOMIC PROFILING OF nICT CELLS: A NEONATAL CD8 LYMPHOCYTE SUBPOPULATION

Background: Premature prelabor rupture of fetal membranes accounts for 30% of all premature births and is associated with detrimental long-term infant outcomes. Understanding PTL, its immunological biology and the role the fetus plays in its trigger may help in preventing a frequent cause of death in newborns. We discovered the presence of a subpopulation of T cells expressing Promyelocytic Leukemia Finger (PLZF+) and RAR related orphan nuclear receptor family (RORγt+) in the umbilical artery, an unexpected cell population in a host occupying a sterile environment (the amniotic space)

Methods: Lymphocytes were isolated by gradient centrifugation from whole blood collected from the umbilical artery and vein of infants delivered by elective uncomplicated cesarean section. Immuno-phenotyping by flow cytometry was performed to identify a subset of CD8+T cells. T cells were stimulated to compare cytokine production and later isolated for single cell mRNA sequencing.

Results: In our repeated approach to detect PLZF+ RORγt+ CD8+T cells (which we refer to as neonatal inflammatory cytotoxic T cells or nicT cells) we found a population of CD8+ T cells that produce TNFα with higher concentration of such cells in the umbilical arterial sample, however we didn't see production of INFγ, Granzyme B or IL-17; additionally we were not able to discern the nicT population on the bases of PLZF+and RORγt+expression as was done previously. We optimized our protocol by using other fixation agents, re-titrating antibodies concentration, and prolonged our activation time by 1.5 hours. We subsequently saw a population of CD161+cells with high PLZF+but did not detect expression of RORγt+T. Our 10X scRNA data and cDNA libraries indicate a specific population clustering with high expression of PLZF+, RORγt+, producing IL-17 and Granzyme B as well as IFNγ.

Conclusion: Our discovery of nicT cells provides important new insight into fetal and neonatal immunobiology. Due to their inflammatory phenotype and localization, these cells may play a previously unidentified role in disorders such as preterm delivery, preeclampsia, and the fetal morbidity/mortalities associated with placental inflammation. They may also be involved in the control of infection or host immune responses to non-replicating antigens. Further we have identified a unique multi-omic profile via single RNA-sequencing. Such data has been used to characterize gene expression of CD8+T cells and surface markers that will help in further understanding the function of this new lineage of cells in the neonate.