Inhibition of autophagy through Beclin1 knockdown diminished the ability of interleukin 36β (IL-36β) to inhibit CD4⁺CD25⁺ regulatory T cells (Tregs). CD4⁺CD25⁺ Tregs isolated from wild-type or Beclin1^+/− mice were stimulated for 48 hours with lipopolysaccharide (LPS) (5 μg/mL) and IL-36β (100 ng/mL). Mice were injected intraperitoneally with IL-36β (1 μg per mouse) and subjected to cecal ligation and puncture (CLP) 2 hours later. (Mice in the sham group were subjected to the same procedure without CLP.) A, B, M, N, The expression levels of forkhead/winged helix transcription factor p3 (Foxp3) and cytotoxic T-lymphocyte antigen 4 in CD4⁺CD25⁺ Tregs were measured at 48 hours using flow cytometry. C–F, Pretreated CD4⁺CD25⁺ Tregs were cocultured with CD4⁺CD25⁻ T cells for 72 hours, and interleukin 10 (IL-10) and transforming growth factor (TGF) β1 levels in the supernatants were determined using enzyme-linked immunosorbent assay (ELISA). K, L, O, The proliferation of CD4+CD25− T cells (responder T cells, Tresp) was measured based on carboxyfluorescein succinimidyl ester (CFSE) staining. G–J, Interferon (IFN) γ, interleukin 2 (IL-2), and interleukin 4 (IL-4) levels in the supernatants were examined by ELISA. *P < .05 and ^†P < .01 (vs LPS or CLP group) (n = 6 per group). P, Beclin1 expression was measured by Western blotting in CD4⁺CD25⁺ Tregs from wild-type mice and Beclin1^+/− mice (n = 3 per group). Abbreviations: APC, allophycocyanin; Cy7, cyanine 7; PBS, phosphate-buffered saline; PE, phycoerythrin.