Biomedical subjects

Sabina Müller

Publications and source records attributed to Sabina Müller.

9 recordsLinked to original sources

PubMed2006

Analysis of peptide/MHC-induced TCR downregulation: deciphering the triggering kinetics.

The interaction of T-lymphocytes with antigen-presenting cells displaying a small number of specific peptide/major histocompatibility complexes results in the downregulation of a large number of T-cell receptors (TCR), suggesting serial TCR triggering. However, the details of TCR downregulation are controversial. In particular, the level of comodulation of nonengaged TCR reported by different authors ranges from essentially none to considerable levels. Here, we address this controversy using complementary experimental and mathematical techniques. We find that TCR downregulation is very rapid during the first 2-4 min after T-cell antigen-presenting cells contact formation. After this phase, TCR downregulation proceeds at a relatively slow rate. Statistical and computational analyses show that this pronounced change in downregulation kinetics is compatible with the notion of initial serial triggering of clustered TCR followed by serial triggering of individual TCR. We further propose a compatible mechanism for concurrent triggering of multiple TCR by a single peptide/major histocompatibility complex. We provide a unified picture of productive TCR engagement and downregulation in which TCR triggering characteristics evolve from an initial cooperative phase to a sustained phase of signal accumulation.

Antigen-Presenting Cells↗

PubMed2005

UV-C light induces raft-associated acid sphingomyelinase and JNK activation and translocation independently on a nuclear signal.

The initiation of UV light-induced signaling in mammalian cells is largely considered to be subsequent to DNA damage. Several studies have also described ceramide (CER), a lipid second messenger, as a major contributor in mediating UV light-induced c-Jun N-terminal kinase (JNK) activation and cell death. It is demonstrated here that UV-C light irradiation of U937 cells results in the activation and translocation of a Zn2+-independent acid sphingomyelinase, leading to CER accumulation in raft microdomains. These CER-enriched rafts aggregate and play a functional role in JNK activation. The observation that UV-C light also induced CER generation and the externalization of acid sphingomyelinase and JNK in human platelets conclusively rules out the involvement of a nuclear signal generated by DNA damage in the initiation of a UV light response, which is generated at the plasma membrane.

Blotting, Western↗

PubMed2005

CD4+ T cells downregulate Bcl-2 in germinal centers.

Germinal centers (GCs) are the main site of T cell-dependent antibody responses. Upon antigen challenge, GCs comprise mostly B cells undergoing proliferation, somatic hypermutation and antigen-affinity selection. GC B cells down-modulate the expression of Bcl-2 protein and are highly sensitive to apoptosis to eliminate autoreactive or low-affinity cells. Bcl-2 is still expressed in a few GC cells, whose identity remains unclear. To address this issue, we examined by confocal microscopy the expression of Bcl-2 by different GC lymphocyte subsets in hyperplastic tonsils. We found that the vast majority of Bcl-2(+) GC cells are T lymphocytes. Conversely, while in the mantle zone and in the interfollicular areas T cells are almost exclusively Bcl-2(+), in the GC, most T lymphocytes are Bcl-2(-). In addition, most of the CD4(+) GC T cells are Bcl-2(-), while nearly 100% of the CD8(+) GC T cells are Bcl-2(+). The Bcl-2 downregulation by both B and CD4(+) T GC cells supports the concept that these two subsets may undergo a selection process in this microenvironment.

CD4-Positive T-Lymphocytes↗

PubMed2005

Immunological synapses are versatile structures enabling selective T cell polarization.

Helper T cells discriminate among different antigen-presenting cells to provide their help in a selective fashion. The molecular mechanisms leading to this exquisite selectivity are still elusive. Here, we demonstrate that immunological synapses are dynamic and adaptable structures allowing T cells to communicate with multiple cells. We show that T cells can form simultaneous immunological synapses with cells presenting different levels of antigenic ligands but eventually polarize toward the strongest stimulus. Remarkably, living T cells form discrete foci of signal transduction of different intensities during the interaction with different antigen-presenting cells and rapidly relocate TCR and Golgi apparatus toward the cell providing the strongest stimulus. Our results illustrate that, although T cell activation requires sustained signaling, T cells are capable of rapid synapse remodeling and swift polarization responses. The combination of sustained signaling with preferential and rapid polarization provides a mechanism for the high sensitivity and selectivity of T cell responses.

Animals↗

PubMed2004

T-cell activation is accompanied by an ubiquitination process occurring at the immunological synapse.

The immunological synapse (IS) is a specialized signaling area formed at the contact site between T-cells and antigen-presenting cells (APC), where sustained engagement and signaling of TCR and accessory molecules occur. A key feature of T-cell antigen recognition is that the process of TCR/peptide-MHC interaction is self-limited by the internalization and degradation of triggered TCR and recruited signaling components. The mechanism of signaling component degradation involves their ubiquitination and targeting for degradation. Yet, the relationship between the ubiquitination process and TCR signaling as well as the cellular localization of TCR-induced ubiquitination are still elusive. In the present work, we visualize for the first time ubiquitination at the TCR signaling area. We show an enrichment of ubiquitin staining in TCR/CD3 caps in T-lymphocytes stimulated by anti-CD3 antibodies. Remarkably, we also show the recruitment of the ubiquitin ligase Cbl-b and a significant ubiquitination at the immunological synapse in antigen-stimulated T-cells. Our results identify the immunological synapse as the cellular area where TCR-induced protein ubiquitination occurs. They imply that the synapse is a specialized site where the activation process is not only triggered, but also controlled via ubiquitination of signaling actors.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Rituximab antiproliferative effect in B-lymphoma cells is associated with acid-sphingomyelinase activation in raft microdomains.

Rituximab is a chimeric human immunoglobulin G1 (IgG1) anti-CD20 monoclonal antibody with significant activity against CD20+ malignant B cells. Rituximab is currently used with success in the treatment of B-cell-derived lymphoid neoplasias either alone or in combination with chemotherapy. However, the predominant mechanism by which rituximab exerts its antitumor properties in vivo remains unknown. In the present study, we demonstrate that in Daudi and RL B-lymphoma cells, rituximab (without cross-linking) used at the saturating dose of 10 microg/mL induced moderate accumulation in G1 phase, growth inhibition, and significant loss in clonogenic potential. However, in these cells, rituximab induced no apoptosis. Furthermore, we observed that treatment with rituximab resulted in a rapid and transient increase in acid-sphingomyelinase (A-SMase) activity and concomitant cellular ceramide (CER) generation in raft microdomains. We also observed that rituximab-treated cells externalized both A-SMase and CER that colocalized with the CD20 receptor. Finally, we present evidence that rituximab-induced growth inhibition may be mediated through a CER-triggered signaling pathway, leading to the induction of cell cycle-dependent kinase inhibitors such as p27Kip1 through a mitogen-activated protein kinase (MAPK)-dependent mechanism.

Antibodies, Monoclonal↗

PubMed2003

Lytic versus stimulatory synapse in cytotoxic T lymphocyte/target cell interaction: manifestation of a dual activation threshold.

Activation of biological functions in T lymphocytes is determined by the molecular dynamics occurring at the T cell/opposing cell interface. In the present study, a central question of cytotoxic T lymphocyte (CTL) biology was studied at the single-cell level: can two distinct activation thresholds for cytotoxicity and cytokine production be explained by intercellular molecular dynamics between CTLs and targets? In this study, we combine morphological approaches with numerical analysis, which allows us to associate specific patterns of calcium mobilization with different biological responses. We show that CTLs selectively activated to cytotoxicity lack a mature immunological synapse while exhibiting a low threshold polarized secretion of lytic granules and spike-like patterns of calcium mobilization. This finding is contrasted by fully activated CTLs, which exhibit a mature immunological synapse and smooth and sustained calcium mobilization. Our results indicate that intercellular molecular dynamics and signaling characteristics allow the definition of two activation thresholds in individual CTLs: one for polarized granule secretion (lytic synapse formation) and the other for cytokine production (stimulatory synapse formation).

Calcium Signaling↗

PubMed2003

Cutting edge: T lymphocyte activation by repeated immunological synapse formation and intermittent signaling.

The activation of biological T cell responses requires prolonged contact with APCs and sustained signaling. We investigated whether signaling must be uninterrupted to commit T cells to cytokine production or whether T cell activation may also result from summation of interrupted signals. Upon periodic addition and removal of a src kinase inhibitor, human CD4(+) T cells destroyed and re-formed immunological synapses while aborting and restarting signal transduction. Remarkably, under these conditions, T cells were eventually activated to IFN-gamma production and the amount of IFN-gamma produced was directly related to the total signaling time despite the repeated interruptions. Our results illustrate that T cell activation does not require a stable immunological synapse and can be achieved by interrupted signaling. It is implied that T cells can add activation signals, possibly collected on multiple APCs.

Antigen Presentation↗

PubMed2002

Cutting edge: TCR engagement and triggering in the absence of large-scale molecular segregation at the T cell-APC contact site.

We investigated the functional role of large-scale molecular segregation at the T cell-APC contact site during T lymphocyte Ag recognition. Inhibition of CD2-CD58 interaction markedly affected segregation of CD2 and CD2AP from CD45. Under these conditions, Ag-induced calcium mobilization, PKC theta; clustering at the immunological synapse, and IFN-gamma production also were inhibited. However, early TCR signaling and T cell polarization toward APCs were unaffected. Our results indicate that the "raison d'être" of a large-scale segregation of surface molecules and intracellular enzymes and adapters, in Ag-stimulated T cells, is to reinforce the assembly of the signal transduction cascade rather than favor TCR engagement and triggering.

Adaptor Proteins, Signal Transducing↗