Role of actin dependent nuclear deformation in regulating early gene expression

The nucleus of a living cell is constantly undergoing changes in shape and size as a result of various mechanical forces in physiology. These changes correlate with alterations in gene expression, however it is unclear whether nuclear deformation alone is sufficient to elicit these alterations. We used T-cell activation as a model system to test the coupling between nuclear deformation (elongation) and gene expression. Naïve T-cell activation with surrogate antigens resulted in actin dependent nuclear elongation. This was accompanied with Erk and NF-κB signaling to the nucleus to induce CD69 expression. Importantly, inhibiting actin polymerization abolished both nuclear elongation and CD69 expression, while inhibiting Erk, NF-κB or microtubule depolymerization only abolished expression but not elongation. Immobilization of antigen-coated beads, under conditions where actin polymerization was inhibited, rescued both nuclear elongation and CD69 expression. In addition, fibroblast cells plated on fibronectin micropatterns of different sizes showed correlation between nuclear shape index and tenascin C expression. Upon inhibiting the signaling intermediate Erk, tenascin C expression was down regulated although the nuclear shape index remained unaltered. Our results highlight the importance of specific signaling intermediates accompanied with nuclear deformation in the modulation of cellular genomic programs

A key control point in the T cell response to chronic infection and neoplasia: FOXO1.

T cells able to control neoplasia or chronic infections display a signature gene expression profile similar or identical to that of central memory T cells. These cells have qualities of self-renewal and a plasticity that allow them to repeatedly undergo activation (growth, proliferation, and differentiation), followed by quiescence. It is these qualities that define the ability of T cells to establish an equilibrium with chronic infectious agents, and also preserve the ability of T cells to be re-activated (by checkpoint therapy) in response to malignant cancers. Here we describe distinctions between the forms of inhibition mediated by tumors and persistent viruses, we review the properties of T cells associated with long-term immunity, and we identify the transcription factor, FOXO1, as the control point for a program of gene expression that allows CD8+ T cells to undergo serial reactivation and self-renewal

Distinct Spatial and Molecular Features of Notch Pathway Assembly in Regulatory T Cells

Variations in the spatial localization of signaling components and crosstalk among signaling cascades are mechanisms through which diversity in signaling networks is generated. The receptor Notch provides an example of regulation by spatial localization: In the canonical Notch signaling pathway, Notch is cleaved to produce the Notch intracellular domain (NICD, also known as NIC), which translocates to the nucleus to regulate gene expression. We describe a T cell receptor-dependent, non-nuclear distribution and function of the processed receptor Notch, which was associated with the improved survival of regulatory T cells (T-regs) in vitro and in vivo and was compromised by T cell-specific deletion of Notch1. Unlike a nuclear-restricted mutant of NICD, mutant NICD that underwent nuclear export or was targeted to the plasma membrane protected Notch1(-/-) T-regs from apoptosis induced by nutrient deprivation and oxidative stress. Notch signaling integrated with phosphatidylinositol 3-kinase signaling and mammalian target of rapamycin complex 2 (mTORC2) for this cell survival function. Biochemical and imaging approaches revealed a membrane-proximal complex containing NICD and the mTORC2 component Rictor, and this complex was stabilized by specific interactions with the Notch ligand Delta-like-1 and mediated the survival of T-regs. Together, our evidence for the spatial control of Notch and the crosstalk of Notch signaling with other pathways reveals coupling between the localization of Notch and diverse intracellular signaling pathways

Apoptotic programs are determined during lineage commitment of CD4(+) T effectors: Selective regulation of T effector-memory apoptosis by inducible nitric oxide synthase

Lineage-committed T effectors generated in response to Ag during the inflammatory phase are destined to die during termination of the immune response. We present evidence to suggest that molecular signatures of lineage commitment are reflected in apoptotic cascades activated in CD4(+) T effectors. Exemplifying this, ablation of inducible NO synthase (iNOS) protected effector-memory T (TEM) cells, but not T-Naive or central-memory T cells, activated in vitro, from apoptosis triggered by cytokine deprivation. Furthermore, attrition of T effectors generated in the secondary, but not the primary, response to Ag was substantially reduced in mice, which received iNOS inhibitors. Distinct patterns of iNOS expression were revealed in wild-type TEM effectors undergoing apoptosis, and ablation of iNOS protein in primary and TEM wild-type effectors confirmed observations made in iNOS(-/-) cells. Describing molecular correlates of this dependence, mitochondrial damage, activation of the protein Bax, and release from mitochondria of the apoptosis-inducing factor were selectively abrogated in iNOS(-/-) TEM effectors. Suggesting that iNOS dependence was linked to the functional identity of T cell subsets, both iNOS induction and apoptosis were compromised in IFN-gamma(-/-) TEM effectors, which mirrored the response patterns of iNOS(-/-) TEM. Collectively, these observations suggest that programs regulating deletion and differentiation are closely integrated and likely encoded during lineage commitment of T effectors. The Journal of Immunology, 2013, 190: 97-105

Rv1218c, an ABC Transporter of Mycobacterium tuberculosis with Implications in Drug Discovery▿ †

Efflux systems are important in determining the efficacy of antibiotics used in the treatment of bacterial infections. In the last decade much attention has been paid to studying the efflux pumps of mycobacteria. New classes of compounds are under investigation for development into potential candidate drugs for the treatment of tuberculosis. Quite often, these have poor bactericidal activities but exhibit excellent target (biochemical) inhibition. Microarray studies conducted in our laboratories for deciphering the mode of action of experimental drugs revealed the presence of putative ABC transporters. Among these transporters, Rv1218c was chosen for studying its physiological relevance in mediating efflux in Mycobacterium tuberculosis. A ΔRv1218c mutant of M. tuberculosis displayed a 4- to 8-fold increase in the inhibitory and bactericidal potency for different classes of compounds. The MICs and MBCs were reversed to wild-type values when the full-length Rv1218c gene was reintroduced into the ΔRv1218c mutant on a multicopy plasmid. Most of the compound classes had significantly better bactericidal activity in the ΔRv1218c mutant than in the wild-type H37Rv, suggesting the involvement of Rv1218c gene product in effluxing these compounds from M. tuberculosis. The implication of these findings on tuberculosis drug discovery is discussed

miR-15/16 clusters restrict effector Treg cell differentiation and function.

Effector regulatory T cells (eTregs) exhibit distinct homeostatic properties and superior suppressor capacities pivotal for controlling immune responses mediated by their conventional T cell counterpart. While the role of microRNAs (miRNAs) in Tregs has been well-established, how miRNAs regulate eTregs remains poorly understood. Here, we demonstrate that miR-15/16 clusters act as key regulators in limiting eTreg responses. Loss of miR-15/16 clusters leads to increased eTreg frequencies with enhanced suppressor function. Consequently, mice with Treg-specific ablation of miR-15/16 clusters display attenuated immune responses during neuroinflammation and upon both infectious and non-infectious challenges. Mechanistically, miR-15/16 clusters exert their regulatory effect in part through repressing IRF4, a transcription factor essential for eTreg differentiation and function. Moreover, miR-15/16 clusters also directly target neuritin, an IRF4-dependent molecule, known for its role in Treg-mediated regulation of plasma cell responses. Together, we identify an miRNA family that controls an important Treg subset and further demonstrate that eTreg responses are tightly regulated at both transcriptional and posttranscriptional levels

Nuclear and cellular remodeling accompany T-cell activation.

<p>a) Representative time-lapse images of T-cells during activation. Green- H2B EGFP nucleus; black- antigen-coated beads. Scale bar 5 µm. b) Schematic representation of experimental design. c) XY trajectory of the centroid of a bead and cell nucleus in M− and M+ conditions. d) Distance between the centroids of a bead and a cell nucleus in M− and M+ conditions.</p

Role for Erk and NF-κB in nuclear elongation and CD69 expression.

<p>a) Normalized histogram showing C_L/C_S values in activated T-cells in M− and M+ conditions in control and cells treated with U0126 or IKK2 inhibitors (n = 200). The red line is used as a cut-off that marks round cells. b) Fraction of cells positive for CD69 in control or in presence of U0126 or IKK2 inhibitors. **p<0.005. Statistical significance is calculated with respect to control samples (M− or M+). c) Fold-change in levels of pERK in T-cells in control (M− and M+) and cytoD treated (M− and M+) conditions (N = 3, mean±s.d.). Inset- representative Western blot for pErk (top) and tubulin (bottom). Lane 1: untreated; 2: M−; 3: M-cytoD+; 4: untreated; 5: M+; 6: M+cytoD+. Statistical significance is calculated for cytoD treated samples with respect to their corresponding control. *p<0.05; **p<0.005. c) Fold change in levels of phosphorylated p130Cas in M− and M+ conditions (N = 3, mean ±s.d.). Inset- representative Western blot for phosphorylated p130Cas (top) and tubulin (bottom). lane 1: untreated; 2: M−; 3: M+. Statistical significance is calculated with respect to M−. **p<0.005.</p