CytoBinning: Immunological insights from multi-dimensional data

New cytometric techniques continue to push the boundaries of multi-parameter quantitative data acquisition at the single-cell level particularly in immunology and medicine. Sophisticated analysis methods for such ever higher dimensional datasets are rapidly emerging, with advanced data representations and dimensional reduction approaches. However, these are not yet standardized and clinical scientists and cell biologists are not yet experienced in their interpretation. More fundamentally their range of statistical validity is not yet fully established. We therefore propose a new method for the automated and unbiased analysis of high-dimensional single cell datasets that is simple and robust, with the goal of reducing this complex information into a familiar 2D scatter plot representation that is of immediate utility to a range of biomedical and clinical settings. Using publicly available flow cytometry and mass cytometry datasets we demonstrate that this method (termed CytoBinning), recapitulates the results of traditional manual cytometric analyses and leads to new and testable hypotheses

Importance of the difference in surface pressures of the cell membrane in doxorubicin resistant cells that do not express Pgp and ABCG2

P-glycoprotein (Pgp) represents the archetypal mechanism of drug resistance. But Pgp alone cannot expel drugs. A small but growing body of works has demonstrated that the membrane biophysical properties are central to Pgp-mediated drug resistance. For example, a change in the membrane surface pressure is expected to support drug–Pgp interaction. An interesting aspect from these models is that under specific conditions, the membrane is predicted to take over Pgp concerning the mechanism of drug resistance especially when the surface pressure is high enough, at which point drugs remain physically blocked at the membrane level. However it remains to be determined experimentally whether the membrane itself could, on its own, affect drug entry into cells that have been selected by a low concentration of drug and that do not express transporters. We demonstrate here that in the case of the drug doxorubicin, alteration of the surface pressure of membrane leaflets drive drug resistance

The Necrotic Signal Induced by Mycophenolic Acid Overcomes Apoptosis-Resistance in Tumor Cells

The amount of inosine monophosphate dehydrogenase (IMPDH), a pivotal enzyme for the biosynthesis of the guanosine tri-phosphate (GTP), is frequently increased in tumor cells. The anti-viral agent ribavirin and the immunosuppressant mycophenolic acid (MPA) are potent inhibitors of IMPDH. We recently showed that IMPDH inhibition led to a necrotic signal requiring the activation of Cdc42.Herein, we strengthened the essential role played by this small GTPase in the necrotic signal by silencing Cdc42 and by the ectopic expression of a constitutive active mutant of Cdc42. Since resistance to apoptosis is an essential step for the tumorigenesis process, we next examined the effect of the MPA–mediated necrotic signal on different tumor cells demonstrating various mechanisms of resistance to apoptosis (Bcl2-, HSP70-, Lyn-, BCR-ABL–overexpressing cells). All tested cells remained sensitive to MPA–mediated necrotic signal. Furthermore, inhibition of IMPDH activity in Chronic Lymphocytic Leukemia cells was significantly more efficient at eliminating malignant cells than apoptotic inducers.These findings indicate that necrosis and apoptosis are split signals that share few if any common hub of signaling. In addition, the necrotic signaling pathway induced by depletion of the cellular amount of GTP/GDP would be of great interest to eliminate apoptotic-resistant tumor cells

Exposed Hydrophobic Residues in Human Immunodeficiency Virus Type 1 Vpr Helix-1 Are Important for Cell Cycle Arrest and Cell Death

The human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein R (Vpr) is a major determinant for virus-induced G2/M cell cycle arrest and cytopathicity. Vpr is thought to perform these functions through the interaction with partner proteins. The NMR structure of Vpr revealed solvent exposed hydrophobic amino acids along helices 1 and 3 of Vpr, which could be putative protein binding domains. We previously showed that the hydrophobic patch along helix-3 was important for G2/M blockade and cytopathicity. Mutations of the exposed hydrophobic residues along helix-1 were found to reduce Vpr-induced cell cycle arrest and cell death as well. The levels of toxicity during virion delivery of Vpr correlated with G2/M arrest. Thus, the exposed hydrophobic amino acids in the amino-terminal helix-1 are important for the cell cycle arrest and cytopathicity functions of Vpr

The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway

Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca2+/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells

ITK is a magnesium sensor during T cell activation

Abstract Interleukin-2-Inductible T-cell Kinase (ITK), a member of the TEC family of kinases, serves a vital role in the activation and differentiation of T-cells. Following stimulation of the T cell receptor (TCR) and phosphorylation by LCK, ITK phosphorylates PLCy1 and initiates a transduction pathway that results in the release of intracellular Ca2+ stores and the transcription of IL-2. Although ITK’s role in this signal transduction pathway is known, there is still much to be characterized about the regulation of ITK activity. Previous studies have indicated that Mg2+ serves a key role as a second messenger in T lymphocyte activation and that attenuated intracellular Mg2+ levels inhibit the phosphorylation of PLCy1 and impair Ca flux. We propose that ITK serves as a sensor for the Mg2+ flux required for optimal T cell activation. Kinase assays show that ITK activity is enhanced by Mg2+ and suggest a model where two Mg2+ ions occupy the catalytic site. Molecular modeling also suggests that ITK possesses two Mg2+ binding sites in the catalytic domain. We are currently studying the effect of Mg2+binding on ITK kinase activity via site directed mutagenesis and are in the process of generating crystal structures of ITK complexed with Mg2+. Finally, we propose that this ‘second site’ Mg2+ is a general feature of kinases and may highlight the importance of divalent cations in kinase regulation.</jats:p