Autophagy plays important roles in metabolism, differentiation, and survival in T cells. TNFAIP3/A20 is a ubiquitin-editing enzyme that is thought to be a negative regulator of autophagy in cell lines. However, the role of TNFAIP3 in autophagy remains unclear. To determine whether TNFAIP3 regulates autophagy in CD4 T cells, we first analyzed Tnfaip3-deficient naïve CD4 T cells in vitro. We demonstrated that Tnfaip3-deficient CD4 T cells exhibited reduced MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) puncta formation, increased mitochondrial content, and exaggerated reactive oxygen species (ROS) production. These results indicate that TNFAIP3 promotes autophagy after T cell receptor (TCR) stimulation in CD4 T cells. We then investigated the mechanism by which TNFAIP3 promotes autophagy signaling. We found that TNFAIP3 bound to the MTOR (mechanistic target of rapamycin) complex and that Tnfaip3-deficient cells displayed enhanced ubiquitination of the MTOR complex and MTOR activity. To confirm the effects of enhanced MTOR activity in Tnfaip3-deficient cells, we analyzed cell survival following treatment with Torin1, an MTOR inhibitor. Tnfaip3-deficient CD4 T cells exhibited fewer cell numbers than the control cells in vitro and in vivo. In addition, the impaired survival of Tnfaip3-deficient cells was ameliorated with Torin1 treatment in vitro and in vivo. The effect of Torin1 was abolished by Atg5 deficiency. Thus, enhanced MTOR activity regulates the survival of Tnfaip3-deficient CD4 T cells. Taken together, our findings illustrate that TNFAIP3 restricts MTOR signaling and promotes autophagy, providing new insight into the manner in which MTOR and autophagy regulate survival in CD4 T cells.
Keywords: 4-OHT, 4-hydroxytamoxifen; ACTB/bACT, actin, β; AKT, v-akt murine thymoma viral oncogene homolog; ATG, autophagy related; ATG5; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BCL10, B-cell CLL/lymphoma 10; BCL2, B-cell CLL/lymphoma 2; CD28, CD28 antigen; CD3E, CD3 antigen, epsilon polypeptide; CD4; CD44, CD44 antigen; CD69, CD69 antigen; CHX, cycloheximide; EIF4EBP1, eukaryotic translation inhibition factor 4E binding protein 1; ESR, estrogen receptor; IFNG, interferon, gamma; IL2, interleukin 2; LPS, lipopolysaccharide; MALT1, MALT1 paracaspase; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MCL1, myeloid cell leukemia 1; MFI, mean fluorescence intensity; MTOR; MTOR, mechanistic target of rapamycin (serine/threonine kinase); NFKB, nuclear factor of kappa light polypeptide gene enhancer in B-cells; PBS, phosphate-buffered saline; PI3K, class I phosphoinositide 3-kinase; PLA, proximity ligation assay; PRKAA/AMPK, protein kinase, AMP-activated; RIPK1, receptor (TNFRSF)-interacting serine-threonine kinase 1; ROS, reactive oxygen species; RPS6KB1, ribosomal protein S6 kinase, polypeptide 1; TCR, T cell receptor; TNFAIP3; TNFAIP3/A20, tumor necrosis factor, α-induced protein 3; TRAF6, TNF receptor-associated factor 6, E3 ubiquitin protein ligase; autophagy; ubiquitin.