Mammalian target of rapamycin (mTOR) plays a crucial role in the control of T cell fate determination; however, the precise regulatory mechanism of the mTOR pathway is not fully understood. We found that T cell-specific deletion of the gene encoding tuberous sclerosis 1 (TSC1), an upstream negative regulator of mTOR, resulted in augmented Th1 and Th17 differentiation and led to severe intestinal inflammation in a colitis model. Conditional Tsc1 deletion in Tregs impaired their suppressive activity and expression of the Treg marker Foxp3 and resulted in increased IL-17 production under inflammatory conditions. A fate-mapping study revealed that Tsc1-null Tregs that lost Foxp3 expression gained a stronger effector-like phenotype compared with Tsc1 -/-Foxp3 + Tregs. Elevated IL-17 production in Tsc1 -/-Treg cells was reversed by in vivo knockdown of the mTOR target S6K1. Moreover, IL-17 production was enhanced by Treg-specific double deletion of Tsc1 and Foxo3a. Collectively, these studies suggest that TSC1 acts as an important checkpoint for maintaining immune homeostasis by regulating cell fate determination.

Various genotoxic agents cause monoubiquitination of NEMO/ IKK;-the regulatory subunit of IKB kinase (IKK) complexin the nucleus. Ubiquitinated NEMO exits from the nucleus and forms a complex with the IKK catalytic subunits IKKA and IKKB, resulting in IKK activation and, ultimately, nuclear factor-KB (NF-KB) activation. Thus, NEMO ubiquitination is a prerequisite for IKK-dependent activation of NF-KB. However, the IKK activation mechanism is unknown and the NEMOubiquitinating E3 enzyme has not been identified. We found that inhibitors of apoptosis protein (IAP) regulate genotoxic stress-induced NF-KB activation at different levels. XIAP mediates activation of the upstream IKK kinase, TAK1, and couples activated TAK1 to the IKK complex. This XIAPdependent event occurs in response to camptotechin or etoposide/VP16; however, XIAP is dispensable for activation of NF-KB by doxorubicin, which engages a MEK-ERK pathway to activate IKK. We also show that cIAP1 mediates NEMO ubiquitination and cIAP2 regulates an event downstream of NEMO ubiquitination. Our study highlights nonredundant cooperative contributions of IAPs to antiapoptotic NF-KB activation by genotoxic signals beyond their classic caspase inhibitory functions. [Cancer Res 2009;69(5):1782-91]

No abstract

NEDD8 (neural precursor cell expressed, developmentally downregulated 8) is a ubiquitin-like molecule whose action on modifying protein substrates is critical in various cellular functions but whose importance in the immune system is not well understood. Here we investigated the role of protein neddylation in regulating T-cell function using an in vivo knockdown technique. We found that reduced expression of Ubc12 in CD4 + T cells led to impaired T-cell receptor/CD28-induced proliferation and cytokine production both in vitro and in vivo, accompanied by reduced Erk activation. These findings were recapitulated by treatment with MLN4924, an inhibitor of NEDD8-activating enzyme. Furthermore, Shc, an adaptor molecule between antigen receptors and the Ras/Erk pathway, was identified as a target for neddylation. Importantly, mice adoptively transferred with Ubc12 knockdown CD4 + T cells showed markedly ameliorated allergic responses. This study thus identifies an important role for protein neddylation in T-cell function, which may serve as a therapeutic target for inflammatory diseases.allergy | signal transduction | posttranslational modification E ngagement of T-cell antigen receptor (TCR) and costimulatory molecules leads to the activation of CD4 + T cells through several signaling pathways, which ultimately induces proliferation, cytokine production, and differentiation into different subsets of T helper type (Th) cells and memory cells (1). Notably, posttranslational modification by ubiquitin and ubiquitin-like proteins (UBLs) has emerged as a critical mechanism regulating T-cell function (2, 3). UBLs compose a diverse group of evolutionarily conserved small proteins, and attachment of different UBLs to a target has different biological consequences (4). Among the UBLs, NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) is covalently attached to lysines in substrate proteins by a series of enzymatic reactions similar to those involved in ubiquitination, including the E1 (Nae1/Uba3) and E2 (Ubc12) reactions (5). The well-characterized substrates of NEDD8 modification are the cullin subunits of cullin-RING ubiquitin E3 ligases, and this modification of cullins is critical for the transfer of ubiquitin from recruited E2-ubiquitin to the substrate (6, 7). The importance of this pathway was further underscored by the recent development of a pharmacologic inhibitor of Nae1, MLN4924, which inhibits cullin neddylation, resulting in substrate accumulation and cancer cell apoptosis by the deregulation of S-phase DNA synthesis, and offers great promise for the treatment of cancer (8-10).In the present study, we investigated whether the NEDD8 pathway is involved in CD4 + T-cell function. Using siRNAmediated depletion of the NEDD8 system in vivo and the NEDD8-activating enzyme inhibitor MLN4924, we found that the NEDD8 pathway is required for TCR-induced proliferation and activation. Interestingly, our results demonstrate that Erk activation by TCR stimulation requires an intact neddylation system. Dif...

Clinical trials are evaluating the efficacy of anti-TIGIT for use as single-agent therapy or in combination with PD-1/PD-L1 blockade. How and whether a TIGIT blockade will synergize with immunotherapies is not clear. Here we show that CD226 lo CD8 + T cells accumulate at the tumor site and have an exhausted phenotype with impaired functionality.In contrast, CD226 hi CD8 + tumor-infiltrating T cells possess greater self-renewal capacity and responsiveness. Anti-TIGIT treatment selectively affects CD226 hi CD8 + T cells by promoting CD226 phosphorylation at tyrosine 322. CD226 agonist antibody-mediated activation of CD226 augments the effect of TIGIT blockade on CD8 + T-cell responses. Finally, mFOLFIRINOX treatment, which increases CD226 hi CD8 + T cells in patients with pancreatic ductal adenocarcinoma, potentiates the effects of TIGIT or PD-1 blockade. Our results implicate CD226 as a predictive biomarker for cancer immunotherapy and suggest that increasing numbers of CD226 hi CD8 + T cells may improve responses to anti-TIGIT therapy.