Mass cytometry: a highly multiplexed single-cell technology for advancing drug development.

Advanced single-cell analysis technologies (e.g., mass cytometry) that help in multiplexing cellular measurements in limited-volume primary samples are critical in bridging discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multiparametric single-cell analysis. Mass cytometers (also known as cytometry by time-of-flight or CyTOF) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of inductively coupled plasma-mass spectrometry. Standard flow cytometry is limited in the number of parameters that can be measured owing to the overlap in signal when detecting fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, which requires minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this overview we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss the emerging and potential applications of CyTOF technology in the pharmaceutical industry, including quantitative and qualitative deep profiling of immune cells and their applications in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies on the basis of phenotype and/or function. We end with a discussion of the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering a drug's pharmacokinetics and pharmacodynamics relationship.

Linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin-2, which acts as a danger signal.

The mechanism by which oxidative stress induces inflammation and vice versa is unclear but is of great importance, being apparently linked to many chronic inflammatory diseases. We show here that inflammatory stimuli induce release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme. Once released, the extracellular PRDX2 acts as a redox-dependent inflammatory mediator, triggering macrophages to produce and release TNF-α. The oxidative coupling of glutathione (GSH) to PRDX2 cysteine residues (i.e., protein glutathionylation) occurs before or during PRDX2 release, a process central to the regulation of immunity. We identified PRDX2 among the glutathionylated proteins released in vitro by LPS-stimulated macrophages using mass spectrometry proteomic methods. Consistent with being part of an inflammatory cascade, we find that PRDX2 then induces TNF-α release. Unlike classical inflammatory cytokines, PRDX2 release does not reflect LPS-mediated induction of mRNA or protein synthesis; instead, PRDX2 is constitutively present in macrophages, mainly in the reduced form, and is released in the oxidized form on LPS stimulation. Release of PRDX2 is also observed in human embryonic kidney cells treated with TNF-α. Importantly, the PRDX2 substrate thioredoxin (TRX) is also released along with PRDX2, enabling an oxidative cascade that can alter the -SH status of surface proteins and thereby facilitate activation via cytokine and Toll-like receptors. Thus, our findings suggest a model in which the release of PRDX2 and TRX from macrophages can modify the redox status of cell surface receptors and enable induction of inflammatory responses. This pathway warrants further exploration as a potential novel therapeutic target for chronic inflammatory diseases.

Glut1-mediated glucose transport regulates HIV infection.

Cell cycle entry is commonly considered to positively regulate HIV-1 infection of CD4 T cells, raising the question as to how quiescent lymphocytes, representing a large portion of the viral reservoir, are infected in vivo. Factors such as the homeostatic cytokine IL-7 have been shown to render quiescent T cells permissive to HIV-1 infection, presumably by transiently stimulating their entry into the cell cycle. However, we show here that at physiological oxygen (O(2)) levels (2-5% O(2) tension in lymphoid organs), IL-7 stimulation generates an environment permissive to HIV-1 infection, despite a significantly attenuated level of cell cycle entry. We identify the IL-7-induced increase in Glut1 expression, resulting in augmented glucose uptake, as a key factor in rendering these T lymphocytes susceptible to HIV-1 infection. HIV-1 infection of human T cells is abrogated either by impairment of Glut1 signal transduction or by siRNA-mediated Glut1 down-regulation. Consistent with this, we show that the susceptibility of human thymocyte subsets to HIV-1 infection correlates with Glut1 expression; single-round infection is markedly higher in the Glut1-expressing double-positive thymocyte population than in any of the Glut1-negative subsets. Thus, our studies reveal the Glut1-mediated metabolic pathway as a critical regulator of HIV-1 infection in human CD4 T cells and thymocytes.

Nerve growth factor: A key local regulator in the pathogenesis of inflammatory arthritis.

OBJECTIVE: The effect of nerve growth factor (NGF) and its receptor (NGFR) in inflammatory diseases is a novel research field. The purpose of this study was to investigate the role of NGF/NGFR in human T cell subpopulations and fibroblast-like synovial cells (FLS) and examine its pathophysiologic significance in psoriatic arthritis (PsA) and rheumatoid arthritis (RA). METHODS: Expression of NGF/NGFR was examined in synovial fluid (SF), FLS, peripheral blood (PB)-derived T cells, and SF-derived T cells from patients with PsA, RA, and osteoarthritis (OA). NGF levels were determined by enzyme-linked immunosorbent assay. NGF-induced T cell/FLS proliferation was examined by MTT assay. Low-affinity (p75)/high-affinity (TrkA) NGFR expression was determined by high-dimensional fluorescence-activated cell sorting. A monochlorobimane assay was used to determine the effect of NGF on T cell survival. RESULTS: Levels of NGF were higher in SF samples from PsA and RA patients as compared to SF samples from OA patients. NGF-induced FLS proliferation was more marked in PsA and RA patients. TrkA was up-regulated on activated SF T cells from PsA (mean ± SD 22 ± 6.2%) and RA (8 ± 1.3%) patients, whereas in SF samples from OA patients, TrkA+CD3+ T cells were not detectable. NGF induced the proliferation of PB T cells, induced the phosphorylation of Akt in activated T cells, and consistent with known pAkt activity, inhibited tumor necrosis factor α-induced cell death in these T cells. CONCLUSION: Based on our findings, we propose a model in which NGF secreted by FLS into PsA and RA synovium promotes the survival of activated autoreactive T cells as well as FLS proliferation. Thus, NGF has the potential to sustain the chronic inflammatory cascades of arthritis of autoimmune origin.

Inherited disorders affecting mitochondrial function are associated with glutathione deficiency and hypocitrullinemia.

Disorders affecting mitochondria, including those that directly affect the respiratory chain function or result from abnormalities in branched amino acid metabolism (organic acidemias), have been shown to be associated with impaired redox balance. Almost all of the evidence underlying this conclusion has been obtained from studies on patient biopsies or animal models. Since the glutathione (iGSH) system provides the main protection against oxidative damage, we hypothesized that untreated oxidative stress in individuals with mitochondrial dysfunction would result in chronic iGSH deficiency. We confirm this hypothesis here in studies using high-dimensional flow cytometry (Hi-D FACS) and biochemical analysis of freshly obtained blood samples from patients with mitochondrial disorders or organic acidemias. T lymphocyte subsets, monocytes and neutrophils from organic acidemia and mitochondrial patients who were not on antioxidant supplements showed low iGSH levels, whereas similar subjects on antioxidant supplements showed normal iGSH. Measures of iROS levels in blood were insufficient to reveal the chronic oxidative stress in untreated patients. Patients with organic acidemias showed elevated plasma protein carbonyls, while plasma samples from all patients tested showed hypocitrullinemia. These findings indicate that measurements of iGSH in leukocytes may be a particularly useful biomarker to detect redox imbalance in mitochondrial disorders and organic acidemias, thus providing a relatively non-invasive means to monitor disease status and response to therapies. Furthermore, studies here suggest that antioxidant therapy may be useful for relieving the chronic oxidative stress that otherwise occurs in patients with mitochondrial dysfunction.

N-Acetylcysteine--a safe antidote for cysteine/glutathione deficiency.

Glutathione (GSH) deficiency is associated with numerous pathological conditions. Administration of N-acetylcysteine (NAC), a cysteine prodrug, replenishes intracellular GSH levels. NAC, best known for its ability to counter acetaminophen toxicity, is a safe, well-tolerated antidote for cysteine/GSH deficiency. NAC has been used successfully to treat GSH deficiency in a wide range of infections, genetic defects and metabolic disorders, including HIV infection and COPD. Over two-thirds of 46 placebo-controlled clinical trials with orally administered NAC have indicated beneficial effects of NAC measured either as trial endpoints or as general measures of improvement in quality of life and well-being of the patients.

Importance of culturing primary lymphocytes at physiological oxygen levels.

Although studies with primary lymphocytes are almost always conducted in CO(2) incubators maintained at atmospheric oxygen levels (atmosO(2); 20%), the physiological oxygen levels (physO(2); 5%) that cells encounter in vivo are 2-4 times lower. We show here that culturing primary T cells at atmosO(2) significantly alters the intracellular redox state (decreases intracellular glutathione, increases oxidized intracellular glutathione), whereas culturing at physO(2) maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore, we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO(2) than at physO(2). This apparently paradoxical finding, we suggest, may be explained by two additional findings with CD3/CD28-stimulated T cells: (i) the intracellular NO (iNO) levels are higher at physO(2) than at atmosO(2); and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO(2) that at atmosO(2). Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo, the lower proliferative T cell responses at physO(2) likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus, we suggest caution in culturing primary lymphocytes at atmosO(2) because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses, particularly after several days in culture.

MYC can induce DNA breaks in vivo and in vitro independent of reactive oxygen species.

MYC overexpression is thought to initiate tumorigenesis by inducing cellular proliferation and growth and to be restrained from causing tumorigenesis by inducing cell cycle arrest, cellular senescence, and/or apoptosis. Here we show that MYC can induce DNA breaks both in vitro and in vivo independent of increased production of reactive oxygen species (ROS). We provide an insight into the specific circumstances under which MYC generates ROS in vitro and propose a possible mechanism. We found that MYC induces DNA double-strand breaks (DSBs) independent of ROS production in murine lymphocytes in vivo as well as in normal human foreskin fibroblasts (NHFs) in vitro in normal (10%) serum, as measured by gammaH2AX staining. However, NHFs cultured in vitro in low serum (0.05%) and/or ambient oxygen saturation resulted in ROS-associated oxidative damage and DNA single-strand breaks (SSBs), as measured by Ape-1 staining. In NHFs cultured in low versus normal serum, MYC induced increased expression of CYP2C9, a gene product well known to be associated with ROS production. Specific inhibition of CYP2C9 by small interfering RNA was shown to partially inhibit MYC-induced ROS production. Hence, MYC overexpression can induce ROS and SSBs under some conditions, but generally induces widespread DSBs in vivo and in vitro independent of ROS production.

Wnt signaling in the thymus is regulated by differential expression of intracellular signaling molecules.

Wnt signaling is essential for T cell development in the thymus, but the stages in which it occurs and the molecular mechanisms underlying Wnt responsiveness have remained elusive. Here we examined Wnt signaling activity in both human and murine thymocyte populations by determining beta-catenin levels, Tcf-reporter activation and expression of Wnt-target genes. We demonstrate that Wnt signaling occurs in all thymocyte subsets, including the more mature populations, but most prominently in the double negative (DN) subsets. This differential sensitivity to Wnt signaling was not caused by differences in the presence of Wnts or Wnt receptors, as these appeared to be expressed at comparable levels in all thymocyte subsets. Rather, it can be explained by high expression of activating signaling molecules in DN cells, e.g., beta-catenin, plakoglobin, and long forms of Tcf-1, and by low levels of inhibitory molecules. By blocking Wnt signaling from the earliest stage onwards using overexpression of Dickkopf, we show that inhibition of the canonical Wnt pathway blocks development at the most immature DN1 stage. Thus, responsiveness to developmental signals can be regulated by differential expression of intracellular mediators rather than by abundance of receptors or ligands.

Culturing at atmospheric oxygen levels impacts lymphocyte function.

To determine whether culturing peripheral blood mononuclear cells at atmospheric oxygen levels skews responses in comparison with culturing lymphocytes at physiologic oxygen levels, we cultured peripheral blood mononuclear cells at 5%, 10%, and atmospheric (20%) gas-phase oxygen for 5 days. We found that incubator oxygen levels influenced lymphocyte proliferation stimulated by two commonly used stimuli: Con A and antibodies that crosslink surface CD3 and CD28 to mimic antigen presentation. In both cases, proliferation increased as gas-phase oxygen levels increased. In contrast, oxygen levels did not influence proliferation stimulated by phytohemagglutinin, another commonly used mitogen. Similarly, oxygen levels did not impact cell viability in unstimulated cultures. Thus, we conclude that the influence of oxygen levels on proliferation depends on the stimulus, and, most importantly from the standpoint of immune responses, culturing cells at atmospheric rather than physiologic oxygen levels results in significantly increased proliferation responses to the CD3/CD28 crosslinking, a proliferation stimulus commonly used to mimic T cell antigen receptor signaling.