Human T Cell Receptor γδ Cells Recognize Endogenous Mevalonate Metabolites in Tumor Cells

T lymphocytes expressing the T cell receptor (TCR)-γδ recognize unknown antigens on tumor cells. Here we identify metabolites of the mevalonate pathway as the tumor ligands that activate TCR-γδ cells. In tumor cells, blockade of hydroxy-methylglutaryl-CoA reductase (HMGR), the rate limiting enzyme of the mevalonate pathway, prevents both accumulation of mevalonate metabolites and recognition by TCR-γδ cells. When metabolite accumulation is induced by overexpressing HMGR or by treatment with nitrogen-containing bisphosphonate drugs, tumor cells derived from many tissues acquire the capacity to stimulate the same TCR-γδ population. Accumulation of mevalonate metabolites in tumor cells is a powerful danger signal that activates the immune response and may represent a novel target of tumor immunotherapy

Antigen recognition and thymic maturation of human TCR Vgamma9-Vdelta2 cells

T cells are divided into two populations according to the type of TCR used for antigen recognition. One population uses a TCR heterodimer, which is composed by the non-covalently associated alpha and beta chains. This TCR recognizes protein and lipid antigens, which are presented by MHC and CD1 antigen-presenting molecules, respectively. A second population uses a TCR heterodimer composed by the gamma and delta chains and recognizes nonpeptidic ligands in the absence of MHC and CD1 restriction. In humans the major population of TCR gd cells uses the Vg9-Vd2 TCR. This is a unique population because it is present only in primates and constitutes >50% of peripheral TCR gd cells. TCR Vg9-Vd2 cells are activated by microbial phosphorylated metabolites and by so far unknown ligands expressed by a group of tumor cells. The principal microbial antigen is (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), an intermediate metabolite generated in 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoids biosynthesis. Despite these cells were described in 1986, many aspects remain unclear, including the nature of the stimulatory ligands present in tumor cells, the mechanisms of their activation during infection, the molecular mechanisms involved in antigen presentation, and the requirements for thymic maturation. In this dissertation we have addressed these important issues using ex vivo cells, biochemical approaches for ligand identification, T cell activation assays and generation of transgenic mice expressing this human TCR. We have identified endogenous metabolites generated in the mevalonate pathway as the tumor ligands which stimulate TCR Vg9-Vd2 lymphocytes. We have found that tumor cells show altered mevalonate pathway which leads to accumulation of intermediate metabolites. This is novel mechanism utilized by the immune system to monitor the metabolic integrity of cells and to react to those which have a dysregulation of this important metabolic pathway. In a second series of studies we have investigated how TCR Vg9-Vd2 cells are activated during bacterial infections. Despite published studies identified HMB-PP as a potent stimulatory ligand in vitro, there was no formal evidence that this compound participates in cell activation during infection. Unexpectedly, we found that HMB-PP is not the major stimulatory ligand during infection and instead endogenous mevalonate metabolites are the stimulatory ligands. We describe how infection modifies the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), which is the key enzyme of the mevalonate pathway, and promotes increased synthesis of stimulatory metabolites. We show that infection induces a transient increase in HMGR protein levels and dephosphorylation, leading to increased enzymatic activity. This alteration occurs already within 1 hour after infection, thus representing a rapid mechanism reacting to infection. Thus, like with recognition of tumor cells, also during infection, the immune system of primates utilizes a mechanism which detects alterations of an important metabolic pathway. We also investigated the mechanisms how mevalonate metabolites traffic within cells. We found that these ligands, which are generated within the cytoplasm, are transported to the cell surface, where they interact with the TCR gd, by the MRP5 transporter. We showed that MRP5-blocking drugs inhibit presentation to TCR gd cells that over expression and knocking down of MRP5 protein increase and inhibit ligand presentation, respectively. These results show that like peptides, which are transported from cytoplasm to the ER through the ABC transporters TAP1 and TAP2, also TCR gd ligands utilize ABC transporters to become immunogenic. We also found that MRP5 is not involved in forming complexes presented to the TCR gd and that other unknown ubiquitous and nonpolymorphic molecules are involved in this process. In the last part of these studies we investigated the requirements for thymic maturation and peripheral expansion of TCR Vg9-Vd2 cells. We generated a transgenic (Tg) mouse model in which T cells express a TCR composed by human Vg9-Vd2 chains. Tg thymocytes express molecules characteristic of partially mature thymocytes together with high levels of Tg TCR. Tg cells do not acquire a mature phenotype and do not exit the thymus in the absence of TCR triggering. However, upon injection of TCR-specific mAbs, Tg thymocytes undergo maturation and colonize peripheral lymphoid organs. Mature Tg T cells remain in the periphery for up to 6 months, with a phenotype of naïve T cells and strongly react to physiological ligands when stimulated by human antigenpresenting cells, which express the restriction element. Thus, Tg T cells expressing the human TCR Vg9-Vd2 resemble TCR ab cells since they also require selection events during thymic maturation

Caspase-4 is required for activation of inflammasomes

IL-1β and IL-18 are crucial regulators of inflammation and immunity. Both cytokines are initially expressed as inactive precursors, which require processing by the protease caspase-1 for biological activity. Caspase-1 itself is activated in different innate immune complexes called inflammasomes. In addition, caspase-1 activity regulates unconventional protein secretion of many other proteins involved in inflammation and repair. Human caspase-4 is a poorly characterized member of the caspase family, which is supposed to be involved in endoplasmic reticulum stress-induced apoptosis. However, its gene is located on the same locus as the caspase-1 gene, which raises the possibility that caspase-4 plays a role in inflammation. In this study, we show that caspase-4 expression is required for UVB-induced activation of proIL-1β and for unconventional protein secretion by skin-derived keratinocytes. These processes require expression of the nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 inflammasome, and caspase-4 physically interacts with its central molecule caspase-1. As the active site of caspase-4 is required for activation of caspase-1, the latter most likely represents a substrate of caspase-4. Caspase-4 expression is also essential for efficient nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 and for absent in melanoma 2 inflammasome-dependent proIL-1β activation in macrophages. These results demonstrate an important role of caspase-4 in inflammation and innate immunity through activation of caspase-1. Therefore, caspase-4 represents a novel target for the treatment of (auto)inflammatory diseases

Propionibacterium acnes promotes Th17 and Th17/Th1 responses in acne patients

Propionibacterium acnes is a Gram-positive commensal bacterium thought to be involved in the pathogenesis of acne vulgaris. Although the ability of P. acnes in the initiation of pro-inflammatory responses is well documented, little is known about adaptive immune responses to this bacterium. The observation that infiltrating immune cells consist mainly of CD4(+) T cells in the perifollicular space of early acne lesions suggests that helper T cells may be involved in immune responses caused by the intra-follicular colonization of P. acnes. A recent report showing that P. acnes can induce IL-17 production by T cells suggests that acne might be a T helper type 17 (Th17)-mediated disease. In line with this, we show in this work that, in addition to IL-17A, both Th1 and Th17 effector cytokines, transcription factors, and chemokine receptors are strongly upregulated in acne lesions. Furthermore, we found that, in addition to Th17, P. acnes can promote mixed Th17/Th1 responses by inducing the concomitant secretion of IL-17A and IFN-γ from specific CD4(+) T cells in vitro. Finally, we show that both P. acnes-specific Th17 and Th17/Th1 cells can be found in the peripheral blood of patients suffering from acne and, at lower frequencies, in healthy individuals. We therefore identified P. acnes-responding Th17/Th1 cells as, to our knowledge, a previously unreported CD4(+) subpopulation involved in inflammatory acne

IL-1β Drives inflammatory responses to propionibacterium acnes in vitro and in vivo

Acne vulgaris is potentially a severe skin disease associated with colonization of the pilo-sebaceous unit by the commensal bacterium Propionibacterium acnes and inflammation. P. acnes is considered to contribute to inflammation in acne, but the pathways involved are unclear. Here we reveal a mechanism that regulates inflammatory responses to P. acnes. We show that IL-1β mRNA and the active processed form of IL-1β are abundant in inflammatory acne lesions. Moreover, we identify P. acnes as a trigger of monocyte-macrophage NLRP3-inflammasome activation, IL-1β processing and secretion that is dependent on phagocytosis, lysosomal destabilization, reactive oxygen species, and cellular K(+) efflux. In mice, inflammation induced by P. acnes is critically dependent on IL-1β and the NLRP3 inflammasome of myeloid cells. These findings show that the commensal P. acnes-by activating the inflammasome-can trigger an innate immune response in the skin, thus establishing the NLRP3-inflammasome and IL-1β as possible therapeutic targets in acne

Peroxisome-derived lipids are self antigens that stimulate invariant natural killer T cells in the thymus

The development and maturation of semi-invariant natural killer T cells (iNKT cells) rely on the recognition of self antigens presented by CD1d restriction molecules in thymus. The nature of the stimulatory thymic self lipids remains elusive. We isolated lipids from thymocytes and found that ether-bonded mono-alkyl glycerophosphates and the precursors and degradation products of plasmalogens stimulated iNKT cells. Synthetic analogs showed high potency in activating thymic and peripheral iNKT cells. Mice deficient in the peroxisomal enzyme glyceronephosphate O-acyltransferase (GNPAT), essential for the synthesis of ether lipids, had significant alteration of the thymic maturation of iNKT cells and fewer iNKT cells in both thymus and peripheral organs, which confirmed the role of ether-bonded lipids as iNKT cell antigens. Thus, peroxisome-derived lipids are nonredundant self antigens required for the generation of a full iNKT cell repertoire