Effect of Atg5 deletion on ILC3 and CD4⁺RORγt⁺ cell proportions

Experimental Design
Core Objective: Flow cytometry was performed to evaluate the effect of RORγt⁺ cell-specific Atg5 knockout (autophagy deficiency) on the proportion of total group 3 innate lymphoid cells (ILC3s), double-negative (DN) ILC3 subsets, and CD4⁺RORγt⁺ T cells among total CD45⁺ immune cells in the intestinal lamina propria of mice with necrotizing enterocolitis (NEC).
Animal Model:Genetically modified mice on a C57BL/6 background were used in this study:
Atg5fl/fl mice: Littermate wild-type control mice with intact Atg5-mediated autophagy function in RORγt⁺ cells
RorccreAtg5fl/fl mice: RORγt⁺ cell-specific conditional Atg5 knockout mice, with autophagy deficiency in RORγt-expressing cells (including the ILC3 lineage and CD4⁺RORγt⁺ T cells)
All mice were subjected to standardized experimental NEC induction prior to sample collection for flow cytometry detection.
Experimental Groups (2 groups total, fully matched to the figure):
Atg5fl/fl group: Control group (NEC-induced Atg5fl/fl mice with autophagy-intact RORγt⁺ cells)
RorccreAtg5fl/fl group: Atg5 knockout group (NEC-induced RorccreAtg5fl/fl mice with RORγt⁺ cell-specific autophagy deficiency)
Detection Targets (fully matched to the figure axes):The proportion of target cell populations among total CD45⁺ immune cells in the intestinal lamina propria, including:
Percentage of total ILC3s among CD45⁺ cells
Percentage of DN ILC3s among CD45⁺ cells
Percentage of CD4⁺RORγt⁺ T cells among CD45⁺ cells
Biological Replicates: 6 biological replicates (individual mice) per group
Statistical Analysis: Unpaired two-tailed Student's t-test was applied for comparisons between the two independent groups. Statistical significance was defined as P < 0.05.
Flow cytometry data were acquired using a CytoFLEX S flow cytometer (Beckman Coulter).

The following fluorochrome-conjugated antibodies were used for cell surface and intranuclear staining in this study: Lineage-FITC, CD45-APC-Cy7, CD4-BV510, CD127-Pacific Blue (PB), CD90.2-PE-Cy7, NKp46-PE, CCR6-APC, and RORγt-PerCP-Cy5.5.