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Graphical AbstractHighlights d LKB1-deficient T reg cells produce Th1 and Th17 cytokines d The loss of LKB1 compromises the mevalonate pathway in T reg cells d Mevalonate or GGPP treatment restores function and stability in LKB1-deficient T reg cells d LKB1-mediated regulation of T reg cells is independent of AMPK SUMMARY The function of regulatory T (T reg ) cells depends on lipid oxidation. However, the molecular mechanism by which T reg cells maintain lipid metabolism after activation remains elusive. Liver kinase B1 (LKB1) acts as a coordinator by linking cellular metabolism to substrate AMP-activated protein kinase (AMPK). We show that deletion of LKB1 in T reg cells exhibited reduced suppressive activity and developed fatal autoimmune inflammation. Mechanistically, LKB1 induced activation of the mevalonate pathway by upregulating mevalonate genes, which was essential for T reg cell functional competency and stability by inducing T reg cell proliferation and suppressing interferon-gamma and interleukin-17A expression independently of AMPK. Furthermore, LKB1 was found to regulate intracellular cholesterol homeostasis and to promote the mevalonate pathway. In agreement, mevalonate and its metabolite geranylgeranyl pyrophosphate inhibited conversion of T reg cells and enhanced survival of LKB1-deficient T reg mice. Thus, LKB1 is a key regulator of lipid metabolism in T reg cells, involved in optimal programming of suppressive activity, immune homeostasis, and tolerance.

The efficacy of combined near-infrared (NIR) and immune therapies for inhibiting tumor growth and recurrence has gained increasing research attention. Regulatory T cells in the tumor microenvironment constitute a major obstacle in achieving robust CD8+ T cell antitumor immunotherapy. In the present study, we designed a photoimmunotherapy-based strategy involving a combination of photothermal and photodynamic therapies, followed by Treg cell suppression, for eliciting an immune response with IR-780- and imatinib-loaded layer-by-layer hybrid nanoparticles.Methods: The layer-by-layer hybrid nanoparticles were prepared through electrostatic interactions. Their photothermal effect, photodynamic effect as well as their effect on inhibiting Treg cells' suppressive function were investigated in vitro and in vivo. Their antitumor effect was evaluated using B16/BL6 and MC-38 tumor-bearing mice.Results: The layer-by-layer hybrid nanoparticles, which were pH-sensitive, enabled the release of IR-780 dye for NIR-induced photothermal and photodynamic effects, and the release of imatinib-loaded glucocorticoid-induced TNF receptor family-related protein/poly(lactic-co-glycolic acid) (GITR-PLGA) nanoparticles to initiate antitumor immunotherapy. The photothermal and photodynamic effects caused by IR-780 under NIR exposure resulted in direct tumor apoptosis/necrosis and the production of tumor-associated antigen, promoted dendritic cell maturation, and enhanced the presentation of tumor-associated antigen to T cells, while the imatinib-loaded GITR-PLGA cores reduced the suppressive function of Treg cells, and consequently activated effective CD8+ T cells towards tumors.Conclusion: With the significant photothermal, photodynamic and immunotherapies, the system successfully eradicated tumor growth, diminished tumor recurrence, and improved survival in vivo. The proposed nanoparticles provide a novel and versatile approach to boost antitumor photoimmunotherapy.

Regulatory CD41 T cells are important for the homeostasis of immune cells, and their absence correlates with autoimmune disorders. However, how the immune system regulates Treg homeostasis remains unclear. We found that IFN-c-deficient-mice had more forkhead box P3 (FOXP3 1) cells than WT mice in all secondary lymphoid organs except the thymus. However, T-bet-or IL-4Ra-deficient mice did not show a similar increase. In vitro differentiation studies showed that conversion of naïve T cells into FOXP3 1 cells (neo-generated inducible Treg (iTreg)) by TGF-b was significantly inhibited by IFN-c in a STAT-1-dependent manner. Moreover, an in vivo adoptive transfer study showed that inhibition of FOXP3 1 iTreg generation by IFN-c was a T-cell autocrine effect. This inhibitory effect of IFN-c on iTreg generation was significantly abrogated after N-acetyl-L-cysteine treatment both in vitro and in vivo, indicating that IFN-c regulation of iTreg generation is dependent on ROS-mediated apoptosis. Therefore, our results suggest that autocrine IFN-c can negatively regulate the neo-generation of FOXP3 1 iTreg through ROSmediated apoptosis in the periphery.

Adiponectin, an adipokine predominantly derived from adipose tissue, exhibits potent antitumor properties in breast cancer cells. However, its mechanisms of action remain elusive. Inflammasomes—intracellular multimeric protein complexes—modulate cancer cell growth in a complicated manner, as well as playing a role in the innate immune system. Herein, we examined the potential role of inflammasomes in the antitumor activity of adiponectin and found that globular adiponectin (gAcrp) significantly suppressed inflammasomes activation in breast cancer cells both in vitro and in vivo conditions, as determined by decreased expression of inflammasomes components, including NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and the apoptosis-associated speck-like protein containing a CARD (ASC), and inhibition of interleukin-1β and caspase-1 activation. Treatment with pharmacological inhibitors of inflammasomes caused decrease in cell viability, apoptosis induction, and G0/G1 cell cycle arrest, suggesting that inflammasomes activation is implicated in the growth of breast cancer cells. In addition, treatment with gAcrp generated essentially similar results to those of inflammasomes inhibitors, further indicating that suppression of breast cancer cell growth by gAcrp is mediated via modulation of inflammasomes. Mechanistically, gAcrp suppressed inflammasomes activation through sestrin2 (SESN2) induction, liver kinase B1 (LKB-1)-dependent AMP-activated protein kinase (AMPK) phosphorylation, and alleviation of endoplasmic reticulum (ER) stress. Taken together, these results demonstrate that gAcrp inhibits growth of breast cancer cells by suppressing inflammasomes activation, at least in part, via SESN2 induction and AMPK activation-dependent mechanisms.

Cross-presentation is a critical process by which antigen is displayed to CD8 T cells to induce tolerance. It is believed that CD8␣ ؉ dendritic cells (DCs) are responsible for cross-presentation, suggesting that the CD8␣ ؉ DC population is capable of inducing both cross-priming and cross-tolerance to antigen. We found that cross-tolerance against intestinal soluble antigen was abrogated in C57BL/6 mice lacking mesenteric lymph nodes (MLNs) and Peyer patches (PPs), whereas mice lacking PPs alone were capable of developing CD8 T-cell tolerance. IntroductionSoluble and cell-associated antigens (Ags) can be presented to CD8 T cells in a process termed cross-presentation. 1 It is believed that this is a major histocompatibility complex (MHC) class I-restricted procedure exclusively mediated by dendritic cells (DCs). Accumulating evidence suggests that cross-presentation provides the immune system with a mechanism by which it prevents autoimmunity and maintains self-tolerance of CD8 T cells against tissue-specific antigens. 1,2 For instance, DCs are continuously sampling self-Ag from tissues and cross-presenting them to CD8 T cells in the draining lymph nodes. Under normal conditions, this leads to clonal deletion of the CD8 T cells by a process termed cross-tolerance. 2 Mouse DCs can be divided phenotypically by the expression of CD8␣, CD11b, CD4, and Gr-1. 3 In the past, CD8␣ ϩ DCs (mainly CD11b Ϫ in spleen) have been referred to as "lymphoid DCs" and CD8␣ Ϫ DCs (mainly CD11b ϩ in spleen) as "myeloid DCs," although this terminology is now thought to be inappropriate. 4 den Haan et al 5 first demonstrated that cell-associated Ag is crosspresented by CD8␣ ϩ DCs but not by CD8␣ Ϫ DCs in the spleen. Moreover, a study showed that this subset of DCs is responsible for the induction of cross-tolerance to tissue-associated self-Ag. 2 Another study reported that intravenous-soluble Ag is also presented by CD8␣ ϩ DCs in the spleen. 6 Of note, a recent study demonstrated that following epidermal infection with herpes simples virus, viral Ag is presented by CD8␣ ϩ DCs but not by Langerhans cells, implicating cross-presentation in such immunity. 7 CD8␣ ϩ DCs have also been reported to be responsible for priming cytotoxic T lymphocyte (CTL) immunity against viral infection via subcutaneous, intravenous, or intranasal routes. 8 Thus, the current paradigm for cross-presentation is that CD8␣ ϩ DCs are responsible for both cross-priming and cross-tolerance to a wide variety of Ags.Under normal circumstances, the mucosal immune system does not induce a protective response against dietary proteins and mucosal self-Ag. 9 In fact, foreign Ags administered by this route have been shown to induce Ag-specific systemic tolerance for CD4 T and CD8 T cells, and B-cell compartments. [10][11][12] Although many studies have investigated the mechanisms of mucosally induced tolerance, 9 the component that regulates Ag-induced tolerance remains largely unclear. In particular, the critical anatomy supporting systemic CD8 T-cell tolerance is not well...

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Background AMP-activated protein kinase (AMPK) is a metabolic sensor that maintains energy homeostasis. AMPK functions as a tumor suppressor in different cancers; however, its role in regulating antitumor immunity, particularly the function of regulatory T cells (Tregs), is poorly defined. Methods AMPKα1fl/flFoxp3YFP-Cre, Foxp3YFP-Cre, Rag1−/−, and C57BL/6 J mice were used for our research. Flow cytometry and cell sorting, western blotting, immuno-precipitation, immuno-fluorescence, glycolysis assay, and qRT-PCR were used to investigate the role of AMPK in suppressing programmed cell death 1 (PD-1) expression and for mechanistic investigation. Results The deletion of the AMPKα1 subunit in Tregs accelerates tumor growth by increasing the expression of PD-1. Metabolically, loss of AMPK in Tregs promotes glycolysis and the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a key enzyme of the mevalonate pathway. Mechanistically, AMPK activates the p38 mitogen-activated protein kinase (MAPK) that phosphorylates glycogen synthase kinase-3β (GSK-3β), inhibiting the expression of PD-1 in Tregs. Conclusion Our study identified an AMPK regulatory mechanism of PD-1 expression via the HMGCR/p38 MAPK/GSK3β signaling pathway. We propose that the AMPK activator can display synergic antitumor effect in murine tumor models, supporting their potential clinical use when combined with anti-PD-1 antibody, anti-CTLA-4 antibody, or a HMGCR inhibitor.