TOP 76 - Spatiotemporal Effects on Quantity and Quality of Regulatory T Cells in Neonatal Hypoxic-Ischemic Encephalopathy

Publication Number: 3808.TOP 76

Stephanie Newman, Children's Hospital of The King's Daughters, Norfolk, VA, United States; Haree Pallera, Children's Hospital of The King's Daughters / Eastern Virginia Medical School, Norfolk, VA, United States; Jeremy D. Moore, Children's Hospital of The King's Daughters, Norfolk, VA, United States; Angela Saadat, eastern virginia medical school, Norfolk, VA, United States; Sai Susmitha, Eastern Virginia Medical School, Norfolk, VA, United States; Asna Sulaiman, Eastern Virginia Medical School, Norfolk, VA, United States; Tushar Shah, Childrens hospital of the kings daughters, Norfolk, VA, United States

Background: Hypoxic-ischemic encephalopathy (HIE) resulting from oxygen deprivation around the time of birth accounts for an estimated 1 million deaths and 42 million disability-adjusted life years globally. Initial injury in the hypoxic is worsened by a burst of inflammatory responses that initiate upon reperfusion, which self-amplify and can lead to chronic neuroinflammation and progressive brain injury.

The role of T lymphocytes in HIE injury remains underexplored. Challenges include that the brain is a highly immune-privileged site with unique, innate-like nuances of neonatal lymphocytes relative to adult lymphocytes. Understanding the role of T cells in HIE is further complicated by sex-dependent immune differences, which are largely due to incomplete inactivation of the X chromosome and consequent overexpression of neural and immune-specific genes.

T regulatory cells (Tregs) are an immunosuppressive subset of T helper cells that become activated after injury. Tregs limit excessive neuroinflammation by regulating microglial activation and infiltration of immune cells into the CNS, and may also have a reparative role by their interaction with oligodendrocytes. The benefits of Tregs in HIE have been shown to be sex-specific, where another research group found that Tregs are higher in female rodent brains and appear to have a beneficial role in mitigating injury, but in males Treg populations are lower and associated with increased secondary neurodegeneration.

Objective: The goal of this study is to characterize changes in quantity and activation status of Tregs and other T lymphocyte subsets over an extended time course from 3, 11, and 90 days after HI injury. Mapping out the time-course of Tregs in neonatal HIE may aid in develop novel targeted therapies to improve outcomes.

Design/Methods: Injury was induced in P11 Wistar rat pups by Vannucci's method ligation of the right carotid artery followed by 50 min at 8% oxygen. Treatment groups received either no treatment/normothermia (NT), therapeutic hypothermia (TH; 6h 31°C rectal temperature) following injury, or no injury or treatment (Sham). The ischemic cerebral hemisphere, spleen and blood were harvested at 3d, 11d, and 90d to capture the hallmark latent, secondary, and tertiary stages of HIE pathology. The abundance of T lymphocyte subsets were measured in homogenates of these tissues by full spectrum flow cytometry (Cytek Aurora) and follow-up analyses with FlowJo software. Data will be analyzed with JMP statistical software and stratified by sex. Data collection is complete, and data analysis will be concluded by January 2026.

Isolation and Quantification of T Lymphocyte Subsets from Neonatal Brain, Spleen and Blood
A) Mice were humanely euthanized using lethal injection of phenobarbital followed by immediate harvesting of tissues. Tissues were passed through 70um filter with ACK lysis buffer to isolate lymphocytes. B) Cell suspensions were incubated with a cocktail of fluorescent-conjugated antibodies to stain for surface and intracellular proteins for identification and activation status. C) Samples were quantified via Cytek Aurora Spectral Flow Cytometer and data analyzed in FlowJo. D) Panel of surface (black), intracellular (blue), and nuclear (gray) markers used to identify and quantify activation status of lymphocyte subsets.

Hypoxic-Ischemic Injury broadly increased subsets of T lymphocytes
Cerebral levels of T lymphocytes, both CD4+ and CD8+, increased in HI-injured rats (NT and TH) from 3 to 11 dpi (A-C). Similarly, levels of transcription factors for Th1 (Tbet), Th2 (GATA3), Treg (Foxp3) and Th17 (IL17a) differentiation all increased in NT and TH but not Sham from 3 to 11 dpi. (D-G) Graphs display means and standard error. N=6 rats per treatment group per harvest point.

Sexual-Dimorphism observed in Cerebral T Helper Cells and Regulatory T Cells in Hypoxic-Ischemic Injury
Cerebral levels of T helper cells (A) and Treg cells (B) increased in over time following injury, and both were higher in injured female rats than male rats. Graphs show means and standard error. *indicates t test p-values <0.05. N=6 rats per treatment group per day, with 6 males and females for each sex.