技术资料

The function of a T cell depends on its subtype and activation state. Here,we show that imaging of the autofluorescence lifetime signals of quiescent and activated T cells can be used to classify the cells. T cells isolated from human peripheral blood and activated in culture using tetrameric antibodies against the surface ligands CD2,CD3 and CD28 showed specific activation-state-dependent patterns of autofluorescence lifetime. Logistic regression models and random forest models classified T cells according to activation state with 97-99{\%} accuracy,and according to activation state (quiescent or activated) and subtype (CD3+CD8+ or CD3+CD4+) with 97{\%} accuracy. Autofluorescence lifetime imaging can be used to non-destructively determine T-cell function. View Publication

Pulmonary sarcoidosis has unknown etiology,a difficult diagnostic procedure and no curative treatment. Extracellular vesicles including exosomes are nano-sized entities released from all cell types. Previous studies of exosomes from bronchoalveolar lavage fluid (BALF) of sarcoidosis patients have revealed pro-inflammatory components and abilities,but cell sources and mechanisms have not been identified. In the current study,we found that BALF exosomes from sarcoidosis patients,but not from healthy individuals,induced a dose-dependent elevation of intracellular IL-1$\beta$ in monocytes. Analyses of supernatants showed that patient exosomes also induced release of IL-1$\beta$,IL-6 and TNF from both PBMCs and enriched monocytes,suggesting that the observed effect is direct on monocytes. The potently chemotactic chemokine CCL2 was induced by exosomes from a subgroup of patients,and in a blocking assay the exosome-induced CCL2 was reduced for 13 out of 19 patients by the asthma drug Montelukast,a cysteinyl leukotriene receptor antagonist. Further,reactive oxygen species generation by PBMCs was induced to a higher degree by patient exosomes compared to healthy exosomes. These findings add to an emerging picture of exosomes as mediators and disseminators of inflammation,and open for further investigations of the link between CCL2 and exosomal leukotrienes in sarcoidosis. View Publication

Hepatic blockade of glucocorticoid receptors (GR) suppresses glucose production and thus decreases circulating glucose levels,but systemic glucocorticoid antagonism can produce adrenal insufficiency and other undesirable side effects. These hepatic and systemic responses might be dissected,leading to liver-selective pharmacology,when a GR antagonist is linked to a bile acid in an appropriate manner. Bile acid conjugation can be accomplished with a minimal loss of binding affinity for GR. The resultant conjugates remain potent in cell-based functional assays. A novel in vivo assay has been developed to simultaneously evaluate both hepatic and systemic GR blockade; this assay has been used to optimize the nature and site of the linker functionality,as well as the choice of the GR antagonist and the bile acid. This optimization led to the identification of A-348441,which reduces glucose levels and improves lipid profiles in an animal model of diabetes. View Publication

Lymphatic endothelial cells (LECs) chemoattract na{\{i}}ve T cells and promote their survival in the lymph nodes and can cross-present antigens to na{\"{i}}ve CD8+ T cells to drive their proliferation despite lacking key costimulatory molecules. However the functional consequence of LEC priming of CD8+ T cells is unknown. Here we show that while many proliferating LEC-educated T cells enter early apoptosis the remainders comprise a long-lived memory subset with transcriptional metabolic and phenotypic features of central memory and stem cell-like memory T cells. In vivo these memory cells preferentially home to lymph nodes and display rapid proliferation and effector differentiation following memory recall and can protect mice against a subsequent bacterial infection. These findings introduce a new immunomodulatory role for LECs in directly generating a memory-like subset of quiescent yet antigen-experienced CD8+ T cells that are long-lived and can rapidly differentiate into effector cells upon inflammatory antigenic challenge.""" View Publication

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however,our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here,we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3,000 covalently liganded cysteines were found on functionally and structurally diverse proteins,including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving the direct functional perturbation and/or degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells and point to electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders. View Publication

Recent studies suggest the effect of radiation is observed not only in irradiated cells but also in adjacent non-irradiated cells (bystander effect),although the mechanism has not yet been fully revealed. This bystander effect may be caused by intercellular communication via a gap junction or by messengers released from irradiated cells,such as reactive oxygen species,nitric oxide,or cytokines. However,an unknown mechanism is also possible in the bystander effect. On the other hand,it is known that extracellular ATP,ADP,uridine 5'-triphosphate (UTP),and uridine 5'-diphosphate (UDP),which are released from cells,act as intercellular signaling molecules by activating purinergic P2X and P2Y receptors (purinergic signaling). Recently,I have suggested these extracellular nucleotides may be novel mediators of a radiation-induced bystander effect,because our recent studies indicated that purinergic signaling is involved in important cellular responses to radiation. Our data indicate that ionizing irradiation causes activation of the transient receptor potential melastatin type 2 (TRPM2) channel,and then ATP is released from cells through the anion channel or connexin43 hemichannel mediated by the activation of a P2X7 receptor. The released nucleotides activate P2Y6 and P2Y12 receptors,which are involved in the DNA damage response after irradiation. Activation of the P2Y6 receptor is also involved in radiation-induced activation of the epithelial growth factor receptor-extracellular signal regulated protein kinase (EGFR-ERK)1/2 pathway and subsequent nuclear translocation of EGFR,which plays a role in DNA repair. Further,the induction of an antioxidant after irradiation is also mediated by the activation of the P2Y receptor. In conclusion,purinergic signaling could play an important role in the protective cellular response to ionizing irradiation. View Publication

Apoptosis,an evolutionarily conserved form of cell suicide,requires specialized machinery. The central component of this machinery is a proteolytic system involving a family of proteases called caspases. These enzymes participate in a cascade that is triggered in response to proapoptotic signals and culminates in cleavage of a set of proteins,resulting in disassembly of the cell. Understanding caspase regulation is intimately linked to the ability to rationally manipulate apoptosis for therapeutic gain. View Publication

The nuclear receptors (NR) retinoid X receptors (RXRs) exert immunomodulatory functions to control inflammation and metabolism via homodimers and heterodimers with several other NRs including retinoic acid receptors. IRX4204 is a novel,highly specific RXR agonist in clinical trials that potently and selectively activates RXR homodimers but not heterodimers. Here,we show that in vivo IRX4204 was compared favorably with FK506 in abrogating acute graft-versus-host disease (GVHD),which was associated with inhibiting allogeneic donor T cell proliferation,reducing T helper 1 differentiation and promoting regulatory T cell (Treg) generation. Recipient IRX4204 treatment reduced intestinal injury and decreased IFN-$\gamma$ and TNF-$\alpha$ serum levels. Transcriptional analysis of donor T cells isolated from intestines of GVHD mice treated with IRX4204 revealed significant decreases in transcripts regulating pro-inflammatory pathways. In vitro,inducible Treg differentiation from na{\{i}}ve CD4+ T cells was enhanced by IRX4204; in vivo IRX4204 increased the conversion of donor Foxp3- T cells into peripheral Foxp3+ Tregs in GVHD mice. Using Foxp3 lineage tracer mice in which both the origin and current FoxP3 expression of Tregs can be tracked we demonstrate that IRX4204 supported Treg stability. Despite favoring Tregs and reducing Th1 differentiation IRX4204-treated recipients maintained graft-versus-leukemia responses against both leukemia and lymphoma cells. Notably IRX4204 reduced in vitro human T cell proliferation and enhanced Treg generation in mixed lymphocyte reaction cultures. Collectively these beneficial effects indicate that targeting RXRs with IRX4204 could be used as a novel approach to prevent acute GVHD in the clinic." View Publication

The caspase family represents a new class of intracellular cysteine proteases with known or suspected roles in cytokine maturation and apoptosis. These enzymes display a preference for Asp in the P1 position of substrates. To clarify differences in the biological roles of the interleukin-1beta converting enzyme (ICE) family proteases,we have examined in detail the specificities beyond the P1 position of caspase-1,-2,-3,-4,-6,and -7 toward minimal length peptide substrates in vitro. We find differences and similarities between the enzymes that suggest a functional subgrouping of the family different from that based on overall sequence alignment. The primary specificities of ICE homologs explain many observed enzyme preferences for macromolecular substrates and can be used to support predictions of their natural function(s). The results also suggest the design of optimal peptidic substrates and inhibitors. View Publication

There is increasing evidence that coronavirus disease 2019 (COVID-19) produces more severe symptoms and higher mortality among men than among women1-5. However,whether immune responses against severe acute respiratory syndrome coronavirus (SARS-CoV-2) differ between sexes,and whether such differences correlate with the sex difference in the disease course of COVID-19,is currently unknown. Here we examined sex differences in viral loads,SARS-CoV-2-specific antibody titres,plasma cytokines and blood-cell phenotyping in patients with moderate COVID-19 who had not received immunomodulatory medications. Male patients had higher plasma levels of innate immune cytokines such as IL-8 and IL-18 along with more robust induction of non-classical monocytes. By contrast,female patients had more robust T cell activation than male patients during SARS-CoV-2 infection. Notably,we found that a poor T cell response negatively correlated with patients' age and was associated with worse disease outcome in male patients,but not in female patients. By contrast,higher levels of innate immune cytokines were associated with worse disease progression in female patients,but not in male patients. These findings provide a possible explanation for the observed sex biases in COVID-19,and provide an important basis for the development of a sex-based approach to the treatment and care of male and female patients with COVID-19. View Publication