Supplementary Material for: Novel Role of p53 in Septic Immunosuppression: Involvement in Loss and Dysfunction of CD4<sup>+</sup> T Lymphocytes

<b><i>Background/Aims:</i></b> Immunosuppression frequently occurs during the development of sepsis and is closely associated with poor outcome. Characteristics of immunosuppressive CD4<sup>+</sup> T lymphocytes in sepsis have been reported to include dramatic cell loss and inactivation. p53 acts as a pivotal transcription factor in regulating cell proliferation and apoptosis, which control tumorigenesis. However, few studies have investigated the universal role of p53 in immune cells, especially in the development of sepsis. <b><i>Methods:</i></b> A mouse model of sepsis was produced by cecal ligation and puncture (CLP), and isolated splenic CD4<sup>+</sup> T cells or Jurkat cells were exposed to lipopolysaccharide (LPS) stimulation <i>in vitro</i>. We used genetic knockout (p53<sup>-/-</sup>) mice or the specific inhibitor pifithrin-α (PFT) to investigate the regulatory mechanisms of p53. Cell proliferation ability was assessed using a Cell Counting Kit-8 assay, and apoptotic cells were stained with annexin V/propidium iodide and then analyzed using a FACScan flow cytometer. Protein and mRNA expression levels were measured by western blotting and real-time PCR, and cytokine levels in culture supernatants were determined by enzyme-linked immunosorbent assay. <b><i>Results:</i></b> Splenic CD4<sup>+</sup> T lymphocytes from CLP mice expressed gradually elevated p53 mRNA and protein levels, which resulted in extracellular regulated protein kinase 1/2 inactivation and expression of apoptotic molecules. Specific inhibition of p53 by PFT or genetic knockout (p53<sup>-/-</sup>) maintained CD4<sup>+</sup> T lymphocyte homeostasis, as indicated by protection from cell loss and restoration of immune function. A medium dose of PFT improved the survival rate of mice, while mortality rate showed only a slight improvement in p53<sup>-/-</sup> mice compared with wild-type mice. The <i>in vitro</i> responses to LPS were consistent with these results, and upregulation of p53 clearly affected the proliferation, apoptosis, and immune dysfunction of CD4<sup>+</sup> T lymphocytes. In addition, we confirmed the regulatory effect of p53 in Jurkat cells, and inhibition of p53 by either inhibition or short hairpin RNA transduction markedly protected cells from LPS stimulation. <b><i>Conclusion:</i></b> Elevation of p53 in T lymphocytes during sepsis or endotoxin challenge might be responsible for inhibiting cell proliferation and enhancing both apoptosis and immune dysfunction of T cells.