Transcriptomic profiling of macrophages that phagocytosed apoptotic Treg or conventional CD4⁺ T (Tconv) cells under different polarization conditions [RNA-Seq]

Macrophage efferocytosis plays a central role in the regulation of inflammation and tissue homeostasis. Regulatory T (Treg) cells are known to influence macrophage function, but the impact of macrophage phagocytosis of apoptotic Treg cells on macrophage polarization remains poorly understood. In this study, bone marrow–derived macrophages (BMDMs) were co-cultured with apoptotic Treg cells or conventional CD4⁺ T (Tconv) cells. Apoptosis of T cells was induced by ATP/NAD⁺ treatment, and CD27 downregulation was used as an indicator of sensitivity to ATP/NAD⁺-mediated cell death. Treg and Tconv cells were labeled with anti-CD4 antibodies conjugated to BV421 or Alexa647 to enable detection of phagocytosis. BMDMs and apoptotic T cells were co-cultured under three conditions: non-polarizing conditions, IFN-γ stimulation, or IL-10 stimulation. After 48 hours of co-culture, macrophages were sorted based on phagocytosis status into three populations: macrophages that had phagocytosed Treg cells, macrophages that had phagocytosed Tconv cells, and macrophages that had not phagocytosed T cells. RNA sequencing was then performed to characterize transcriptional changes associated with phagocytosis of different T cell subsets and polarization conditions.