Comparison of Fas(Apo-1/CD95)- and perforin-mediated cytotoxicity in primary T lymphocytes

Cytolytic T lymphocytes kill target cells by two independent cytolytic mechanisms. One pathway depends on the polarized secretion of granule-stored proteins including perforin and granzymes, causing target cell death through membrane and DNA damage. The second cytolytic effector system relies on the interaction of the Fas ligand (Fasl) on the effector cell with its receptor (Fas) on the target cell, leading to apoptotic cell death. Using mixed lymphocyte culture (MLC)-derived primary T lymphocytes of perforin-knockout and gld (with non-functional FasL) mice, the molecular basis of the two killing mechanisms was compared. The activity of both pathways was dependent on extracellular Ca2+. Incubation of MLC-stimulated primary T cells with protein synthesis inhibitors prior to TCR triggering impaired FasL cell surface expression and abolished cytolytic activity, although the cells exhibited an intracellular pool of FasL. The perforin-dependent mechanism induced cell death more rapidly, although both pathways ultimately showed similar killing efficiencies. Both pathways induced comparable levels of DNA degradation, but Fas-induced membrane damage was less pronounced. We conclude that upon TCR triggering FasL may be recruited in part from pre-existing intracellular stores. However, efficient induction of target cell death still depends on the continuous biosynthesis of FasL molecules

T-cell intracellular antigens in health and disease

T-cell intracellular antigen 1 (TIA1) and TIA1-related/like protein (TIAR/TIAL1) are 2 proteins discovered in 1991 as components of cytotoxic T lymphocyte granules. They act in the nucleus as regulators of transcription and pre-mRNA splicing. In the cytoplasm, TIA1 and TIAR regulate and/or modulate the location, stability and/or translation of mRNAs. As knowledge of the different genes regulated by these proteins and the cellular/biological programs in which they are involved increases, it is evident that these antigens are key players in human physiology and pathology. This review will discuss the latest developments in the field, with physiopathological relevance, that point to novel roles for these regulators in the molecular and cell biology of higher eukaryotes.Ministry Economic Affairs and Competitiveness through FEDER funds (BFU2008–00354, BFU2011–29653 and BFU2014–57735-R). The CBMSO receives an institutional grant from Fundación Ramón Areces.Peer Reviewe

Characterization of the non-functional Fas ligand of gld mice

Mice homozygous for either the gld or lpr mutation develop autoimmune diseases and progressive lymphadenopathy. The lpr mutation is characterized by the absence of functional Fas, whereas gld mice exhibit an inactive FasL due to a point mutation proximal to the extracellular C-terminus. The structural repercussions of this amino acid substitution remain unknown. Here we report that FasL is expressed at similar levels on the surface of activated T lymphocytes from gld and wild-type mice. Using a polyclonal anti-FasL antibody, indistinguishable amounts of a 40 kDa protein are detected in both gld and wild-type splenocytes. The molecular model of FasL, based on the known structure of TNF-alpha, predicts that the Phe --> Leu gld mutation is located at the protomer interface which is close to the FasR interaction site. We conclude that the gld mutation allows normal FasL biosynthesis, surface expression and oligomerization, but induces structural alterations to the Fas binding region leading to the phenotypic changes observed

A Model for the Interplay of Receptor Recycling and Receptor-Mediated Contact in T Cells

Orientation of organelles inside T cells (TC) toward antigen-presenting cells (APC) ensures that the immune response is properly directed, but the orientation mechanisms remain largely unknown. Structural dynamics of TC are coupled to dynamics of T-cell receptor (TCR), which recognizes antigen on the APC surface. Engagement of the TCR triggers its internalization followed by delayed polarized recycling to the plasma membrane through the submembrane recycling compartment (RC), which organelle shares intracellular location with the TC effector apparatus. TCR engagement also triggers TC-APC interface expansion enabling further receptor engagement. To analyze the interplay of the cell-cell contact and receptor dynamics, we constructed a new numerical model. The new model displays the experimentally observed selective stabilization of the contact initiated next to the RC, and only transient formation of contact diametrically opposed to the RC. In the general case wherein the TC-APC contact is initiated in an arbitrary orientation to the RC, the modeling predicts that the contact dynamics and receptor recycling can interact, resulting effectively in migration of the contact to the TC surface domain adjacent to the submembrane RC. Using three-dimensional live-cell confocal microscopy, we obtain data consistent with this unexpected behavior. We conclude that a TC can stabilize its contact with an APC by aligning it with the polarized intracellular traffic of TCR. The results also suggest that the orientation of TC organelles, such as the RC and the effector apparatus, toward the APC can be achieved without any intracellular translocation of the organelles

Expression of FAP-1 by human colon adenocarcinoma: implication for resistance against Fas-mediated apoptosis in cancer

Although colon carcinoma cells express Fas receptors, they are resistant to Fas-mediated apoptosis. Defects within the intracellular Fas signal transduction may be responsible. We investigated whether the Fas-associated phosphatase-1 (FAP-1), an inhibitor of Fas signal transduction, contributed to this resistance in colon carcinomas. In vivo, apoptosis of cancer cells was detected in situ using terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling ( TUNEL). FAP-1, FasR, and Fas ligand (FasL) were detected using immunohistochemistry. In vitro, colon carcinoma cells were primarily cultured, and their sensitivity to Fas-mediated apoptosis was evaluated by treatment with agonistic anti-FasR CH11 IgM monoclonal antibody in the presence or absence of synthetic Ac-SLV (serine-leucine-valine) tripeptide. Fas-associated phosphatase-1 expression was detected in 20 out of 28 colon adenocarcinomas. In vivo, a positive correlation between the percentage of apoptotic tumour cells and the number of FasL-positive tumour infiltrating lymphocytes was observed in FAP-1 negative cancers, but not in FAP-1-positive ones. Primarily cultured colon cancer cells, which were refractory to CH-11-induced apoptosis, had higher expression of FAP-1 on protein and mRNA levels than the sensitive group. Resistance to Fas-mediated apoptosis in tumour cells could be abolished by Ac-SLV tripetides. Fas-associated phosphatase-1 expression protects colon cancer cells from Fas-mediated apoptosis, and blockade of FAP-1 and FasR interaction sensitises tumour cells to Fas-dependent apoptosis

An Experimental and Computational Study of Effects of Microtubule Stabilization on T-Cell Polarity

T-killer cells eliminate infected and cancerous cells with precision by positioning their centrosome near the interface (immunological synapse) with the target cell. The mechanism of centrosome positioning has remained controversial, in particular the role of microtubule dynamics in it. We re-examined the issue in the experimental model of Jurkat cells presented with a T cell receptor-binding artificial substrate, which permits controlled stimulation and reproducible measurements. Neither 1-µM taxol nor 100-nM nocodazole inhibited the centrosome positioning at the “synapse” with the biomimetic substrate. At the same time, in micromolar taxol but not in nanomolar nocodazole the centrosome adopted a distinct peripheral rather than the normally central position within the synapse. This effect was reproduced in a computational energy-minimization model that assumed no microtubule dynamics, but only a taxol-induced increase in the length of the microtubules. Together, the experimental and computational results indicate that microtubule dynamics are not essential for the centrosome positioning, but that the fit of the microtubule array in the deformed body of the conjugated T cell is a major factor. The possibility of modulating the T-cell centrosome position with well-studied drugs and of predicting their effects in silico appears attractive for designing anti-cancer and antiviral therapies

Resistance of MLL–AFF1-positive acute lymphoblastic leukemia to tumor necrosis factor-alpha is mediated by S100A6 upregulation

Mixed-lineage leukemia (MLL)–AFF1 (MLL–AF4)-positive acute lymphoblastic leukemia (ALL) is associated with poor prognosis, even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The resistance to graft-versus-leukemia (GVL) effects may be responsible for the poor effect of allo-HSCT on MLL–AFF1-positive ALL. Cytotoxic effector mechanisms mediated by tumor necrosis factor-alpha (TNF-α) was reported to contribute to the GVL effect. We showed that MLL–AFF1-positive ALL cell lines are resistant to TNF-α. To examine the mechanism of resistance to TNF-α of MLL–AFF1-positive leukemia, we focused on S100A6 as a possible factor. Upregulation of S100A6 expression and inhibition of the p53–caspase 8–caspase 3 pathway were observed only in MLL–AFF1-positive ALL cell lines in the presence of TNF-α. The effect of S100A6 on resistance to TNF-α by inhibition of the p53–caspase 8–caspase 3 pathway of MLL–AFF1-positive ALL cell lines were also confirmed by analysis using small interfering RNA against S100A6. This pathway was also confirmed in previously established MLL–AFF1 transgenic mice. These results suggest that MLL–AFF1-positive ALL escapes from TNF-α-mediated apoptosis by upregulation of S100A6 expression, followed by interfering with p53–caspase 8–caspase 3 pathway. These results suggest that S100A6 may be a promising therapeutic target for MLL–AFF1-positive ALL in combination with allo-HSCT

HIV-1 Nef Employs Two Distinct Mechanisms to Modulate Lck Subcellular Localization and TCR Induced Actin Remodeling

The Nef protein acts as critical factor during HIV pathogenesis by increasing HIV replication in vivo via the modulation of host cell vesicle transport and signal transduction processes. Recent studies suggested that Nef alters formation and function of immunological synapses (IS), thereby modulating exogenous T-cell receptor (TCR) stimulation to balance between partial T cell activation required for HIV-1 spread and prevention of activation induced cell death. Alterations of IS function by Nef include interference with cell spreading and actin polymerization upon TCR engagement, a pronounced intracellular accumulation of the Src kinase Lck and its reduced IS recruitment. Here we use a combination of Nef mutagenesis and pharmacological inhibition to analyze the relative contribution of these effects to Nef mediated alterations of IS organization and function on TCR stimulatory surfaces. Inhibition of actin polymerization and IS recruitment of Lck were governed by identical Nef determinants and correlated well with Nef's association with Pak2 kinase activity. In contrast, Nef mediated Lck endosomal accumulation was separable from these effects, occurred independently of Pak2, required integrity of the microtubule rather than the actin filament system and thus represents a distinct Nef activity. Finally, reduction of TCR signal transmission by Nef was linked to altered actin remodeling and Lck IS recruitment but did not require endosomal Lck rerouting. Thus, Nef affects IS function via multiple independent mechanisms to optimize virus replication in the infected host