scholarly journals The Bardet-Biedl syndrome complex component BBS1 controls T cell polarity during immune synapse assembly

2021 ◽  
Author(s):  
Chiara Cassioli ◽  
Anna Onnis ◽  
Francesca Finetti ◽  
Nagaja Capitani ◽  
Jlenia Brunetti ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Trafficking ◽  
Synapse Formation ◽  
Intraflagellar Transport ◽  
Regulatory Subunit ◽  
Bardet Biedl Syndrome ◽  
Immune Synapse ◽  
Microtubule Motor ◽  
Cell Synapse

Components of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are co-opted by the non-ciliated T cell to orchestrate polarized endosome recycling and to sustain signaling during immune synapse formation. Here we have investigated the potential role of BBS1, an essential core component of the Bardet-Biedl syndrome complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We demonstrate that BBS1 allows for centrosome polarization towards the immune synapse. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH, a process that we demonstrate to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that acts upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling.

scholarly journals The Bardet-Biedl Syndrome complex component BBS1 regulates proteasome-dependent F-actin clearance from the centrosome to enable its translocation to the T cell immune synapse

2020 ◽  
Author(s):  
Chiara Cassioli ◽  
Anna Onnis ◽  
Francesca Finetti ◽  
Nagaja Capitani ◽  
Ewoud B Compeer ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Trafficking ◽  
Synapse Formation ◽  
Intraflagellar Transport ◽  
Regulatory Subunit ◽  
Bardet Biedl Syndrome ◽  
Immune Synapse ◽  
Microtubule Motor ◽  
Cell Synapse

ABSTRACTComponents of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are exploited by the non-ciliated T cell to orchestrate polarized endosome recycling to sustain signaling during immune synapse formation. Here we have investigated the potential role of BBS1, an essential core component of the Bardet-Biedl syndrome complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We show that BBS1 allows for centrosome polarization towards the immune synapse by promoting its untethering from the nuclear envelope. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH, a process that we demonstrate to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that acts upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling.

Functional Screening Studies Identify Combinational Activity of PD-L1 and CD200 In Mediating Dysfunctional T Cell Immunological Synapse Formation In Chronic Lymphocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 696-696
Author(s):  
Alan G. Ramsay ◽  
Andrew James Clear ◽  
Alexander Davenport ◽  
Rewas Fatah ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Immune Suppression ◽  
Neutralizing Antibodies ◽  
Synapse Formation ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Immune Synapse ◽  
Cell Synapse

Abstract Abstract 696 The ability of cancer cells to modulate the immune microenvironment is now recognized as an important hallmark of disease pathophysiology. Identifying the molecular mechanisms of cancer immune suppression in the laboratory is key to the design of more effective immunotherapeutic treatment strategies. We previously demonstrated that chronic lymphocytic leukemia (CLL) cells induce alterations in global gene expression profiles in patient CD4 and CD8 T cells, and a profound T cell immunological synapse formation defect that can be reversed with lenalidomide (J Clin Invest. 2005;115(7):1797-1805, and 2008;118(7):2427-2437). Here we used small interfering RNA (siRNA) with a 2-part functional screen to identify key CLL cell molecules inducing T cell immune suppression. siRNA treated tumor cells were cocultured in direct contact with healthy allogeneic T cells for 24 hours, T cells purified from coculture and used in cell conjugation immune synapse assays with superantigen-pulsed third party B cells as antigen-presenting cells (APCs). Confocal microscopy and image analysis software was used to quantify the mean area of T cell F-actin immune synapse formation events from each experimental cell population. Treatment of the CLL cell line MEC-1 with either TNFα, TGFβ, IL-10, or IL-6 siRNA identified no gain in subsequent CD3 T cell immune synapse function compared to control non-targeting siRNA or untreated CLL cells. However, CD200 or programmed death 1 (PD1) ligand 1 (PD-L1, CD274) siRNA treatment significantly enhanced (P < .01) subsequent T cell synapse formation events with APCs (comparable to positive control experiments blocking tumor cell:T cell direct contact with ICAM-1 siRNA, or primary coculture of T cells with allogeneic healthy donor B cells). Primary CLL patient cells (n=10) were treated with individual or pooled neutralizing antibodies, or siRNA, targeting PD-L1, CD200, or cytokines. This analysis revealed that counteracting the combined activity of PD-L1, CD200 and TGFβ exhibited the most pronounced repair of subsequent T cell synapse function compared to control treated tumor cells (P < .01). These data suggest that CLL-released cytokines such as TGFβ contribute to, but are not essential for the T cell synapse defect. We also identified that blocking the T cell receptors PD-1, CD200-R and TGFβ-R1 with neutralizing antibodies prevents CLL inhibitory signaling (P < .01) compared to isotype control IgG treated T cells in contact with tumor cells. We further show that knock-down of PD-L1, CD200 and TGFβ on ex vivo CLL cells prevents inhibitory CD4 and CD8 T cell synapse function compared to control siRNA (P < .01) using the Eμ-TCL1 mouse model of CLL. The addition of lenalidomide (1μM) in ex vivo CLL cell:T cell coculture assays significantly increased (P < .01) subsequent T cell synapse function compared to untreated vehicle control experiments. Flow cytometric analysis identified that lenalidomide down-regulates both CLL expressed PD-L1 and CD200 ligands, and T cell cognate receptor PD1 and CD200R expression during intercellular contact interactions. Moreover, subsequent effector T cell killing function was significantly enhanced (P < .05) following antibody blockade of CLL cell PD-L1 and CD200 with or without lenalidomide treatment during primary coculture with CD8 T cells. We are currently investigating the expression and activity of PD-L1, CD200, and other co-inhibitory molecules in CLL and other haematological and solid malignancies, using patient tissue microarray analysis and confocal co-localization analysis. This work is identifying common inhibitory ligands utilized by tumor cells to suppress T cell synapse function. These results provide important mechanistic insight into immune suppression in CLL and the action of lenalidomide, and identify co-inhibitory ligands as potential immunotherapeutic targets to repair T cell function. Disclosures: Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.

scholarly journals CD33 BiTE ® Construct Mediated Immunological Synapse Formation and Downstream Signaling in T Cells Is Dependent on Expression of Costimulatory Molecules on Target Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2237-2237
Author(s):  
Anetta Marcinek ◽  
Bettina Brauchle ◽  
Gerulf Hänel ◽  
Sonja M Lacher ◽  
Nora Zieger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Synapse Formation ◽  
Costimulatory Molecules ◽  
Target Cells ◽  
Immune Synapse ◽  
Downstream Signaling ◽  
Signaling Receptors

Abstract BiTE ® (Bispecific T-cell Engager) constructs represent a novel immunotherapeutic strategy that recruits T cells against cancer cells independent of their TCR specificity. Currently, two CD33xCD3 BiTE ® antibody constructs (AMG 330 & AMG 673) are being investigated in phase I dose escalation trials in patients with relapsed/refractory Acute Myeloid Leukemia (AML) with early evidence of acceptable safety and anti-leukemic activity (Ravandi et al., ASH 2020; Subklewe et al., EHA 2020). So far, details of BiTE ® mediated T-cell engagement and information on parameters contributing to their efficacy need more investigation. Therefore, we aimed to characterize the interplay between target and effector cells to deepen our mechanistic understanding of BiTE ® construct mediated T-cell engagement. Previously, we have created a novel in vitro model system with murine Ba/F3 cells expressing human (hu) CD33 ± huCD80 ± huCD86 ± huPD-L1 to study T-cell proliferation and cytotoxicity induced by AMG 330. Using that system, we showed that expression of T-cell co-signaling receptors on target cells modulate AMG 330 induced T-cell activity (Marcinek et al., ASH 2018, EHA 2019). Here, we hypothesize that expression of costimulatory molecules impacts BiTE ® mediated immune synapse formation and consecutive downstream signaling in BiTE ® construct activated T cells. To study whether AMG 330 can induce synapse formation and TCR triggering we used a previously described reconstituted T-cell system, which consists of non-immune (HEK) cells introduced with genes encoding the TCR and other proteins (e.g. CD45) required for the regulation of TCR phosphorylation (James et al., Nature 2012). HEK-T cells were incubated with huCD33 transduced RajiB cells in presence of fluorescently labeled AMG 330 or a control BiTE® (cBiTE) construct to allow cell conjugation. A spinning disc confocal microscope system was used to image cells. To pinpoint the role of T-cell co-signaling receptors in immune synapse formation we incubated differentBa/F3 cell constructs or primary AML (pAML) cells with healthy donor T cells in the presence of AMG 330 and analyzed intensity of LFA-1 expression within the synapse using an Imaging Flow Cytometer. Furthermore, we determined phosphorylation of ZAP70, AKT and ERK in conjugated T cells after various time points by phosphoflow cytometry. We observed that AMG 330, in contrast to cBiTE®, induced TCR triggering reflected by exclusion of CD45 from the RajiB-T-cell-interface. Simultaneously clustering of CD33 occurred in AMG 330 induced cell-cell-interfaces (Fig. 1A/B). The percentage of conjugates formed with huCD33 + Ba/F3 cells was significantly higher in constructs expressing huCD86, compared to those expressing no costimulatory antigens or additional huPD-L1 (Mean % in huCD33 + Ba/F3: 2.8 vs. huCD33 + CD86 +.Ba/F3: 4.2 [p=0.0031] vs. huCD33 + huCD86 + PD-L1 + Ba/F3: 2.8 [p=0.0018]). This was accompanied by LFA-1 accumulation within the T-cell-Ba/F3 cell synapse (Mean of MFI in huCD33 + CD86 +.Ba/F3: 10,933 &gt; huCD33 + huCD86 + PD-L1 + Ba/F3: 7,749 &gt; huCD33 + Ba/F3: 7,028). For downstream signaling in T cells after engagement with Ba/F3 cell constructs in the presence of AMG 330, we observed that kinase phosphorylation was highest after 10 minutes in CD86 co-expressing Ba/F3 cells (Mean % of phosphorylation in T-cell conjugates with huCD33 + vs huCD33 + huCD86 + vs huCD33 + CD86 +.PD-L1 + Ba/F3: pERK 40.9 vs 54.3 [p=0.0064] vs 51.2 %; pAKT: 69.1 vs 81.5 [p=0.0642] vs 75.1 %; pZAP70: 6.9 vs 12.2 [p&lt;0.0001] vs 7.7 % [p&lt;0.0001]) (Fig. 1C). Finally, we evaluated if these finding could also be observed in pAML samples. For that, we determined LFA-1 expression intensity within AMG 330-induced pAML-T-cell synapses. We used CD33 + pAML samples with either high CD86 and no PD-L1 expression or vice versa. Comparing synapse formation between these samples, LFA-1 intensity was 4.6-fold higher in the CD86 + PD-L1 - sample compared to the CD86 - PD-L1 + pAML. Taken together, our data unravel molecular mechanisms of BiTE® construct induced immune synapse formation, highlighting the role of costimulatory molecules in this process. They support the notion that T cell co-signaling receptors like CD86 and PD-L1 modulate T-cell response in an early event manner. Prospective analyses in clinical trials are needed to validate the relevance of checkpoint molecule expression on target cells as a potential predictive biomarker for response. Figure 1 Figure 1. Disclosures Brauchle: Adivo: Current Employment. Lacher: Roche: Research Funding. Kischel: Amgen GmbH Munich: Current Employment. von Bergwelt: Roche: Honoraria, Research Funding, Speakers Bureau; Miltenyi: Honoraria, Research Funding, Speakers Bureau; Mologen: Honoraria, Research Funding, Speakers Bureau; Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Research Funding, Speakers Bureau; MSD Sharpe & Dohme: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau. Theurich: Amgen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Buecklein: Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Miltenyi: Research Funding; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Subklewe: Janssen: Consultancy; Seattle Genetics: Consultancy, Research Funding; Roche: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Klinikum der Universität München: Current Employment; Takeda: Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau.

The neutral sphingomyelinase 2 in T cell receptor signaling and polarity

2018 ◽  
Vol 399 (10) ◽  
pp. 1147-1155 ◽  
Author(s):  
Lena Collenburg ◽  
Sibylle Schneider-Schaulies ◽  
Elita Avota
Keyword(s):  
T Cell ◽  
Stress Responses ◽  
Synapse Formation ◽  
Immune Cell ◽  
Cell Receptor ◽  
Immune Synapse ◽  
Neutral Sphingomyelinase ◽  
Cytoskeletal Dynamics ◽  
Cellular Stress Responses

AbstractBy hydrolyzing its substrate sphingomyelin at the cytosolic leaflet of cellular membranes, the neutral sphingomyelinase 2 (NSM2) generates microdomains which serve as docking sites for signaling proteins and thereby, functions to regulate signal relay. This has been particularly studied in cellular stress responses while the regulatory role of this enzyme in the immune cell compartment has only recently emerged. In T cells, phenotypic polarization by co-ordinated cytoskeletal remodeling is central to motility and interaction with endothelial or antigen-presenting cells during tissue recruitment or immune synapse formation, respectively. This review highlights studies adressing the role of NSM2 in T cell polarity in which the enzyme plays a major role in regulating cytoskeletal dynamics.

scholarly journals Mir-181c Modulates T Cell Function By Regulating the Expression of BRK1

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 132-132
Author(s):  
Shok Ping Lim ◽  
Donal McLornan ◽  
Nikolaos Ioannou ◽  
David Darling ◽  
Alan G. Ramsay ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Synapse Formation ◽  
Cell Function ◽  
Immune Synapse ◽  
T Cell Function ◽  
Lamellipodia Formation

Abstract Introduction MicroRNAs (miRNAs) are short endogenous non-coding RNAs consisting of 18-25 nucleotides in length which influence gene expression and play pivotal roles in a diverse range of cellular processes. Aberrant miRNA expression has been implicated in a variety of cancers, including haematological malignancies. The miR-181 family plays a crucial role in haematopoiesis, including megakaryocytic, erythroid and myeloid differentiation and both B and T cell development and differentiation. We therefore focused our study on validating novel downstream targets of miR-181. Methods A novel functional assay utilising an optimised 3'UTR enriched library and a dual selection strategy (Gäken et al., 2012) was performed to identify biologically relevant targets of miR-181c. BRK1 (BRICK1, SCAR/WAVE Actin Nucleating Complex Subunit) was identified as a potential target and validation was performed by quantitative real time PCR and western blot analysis. Given the potential role of BRK1 in the Wiskott-Aldrich Syndrome Protein Family Verprolin-Homologous Protein-2 (WAVE2) complex and actin polymerisation in T cells, we investigated the influence of the miR-181c-BRK1 axis on T cell function. Knockdown of BRK1, using short hairpin RNA (shRNA) lentiviral vectors, and overexpression of miR-181c, via transfection with miR-181c expression vectors, were performed in Jurkat and primary T cells. T cell activation was examined by measurement of CD69 and CD154 expression and actin polymerisation was quantified by total cellular F-actin content. Immune synapse formation was studied by conjugate formation between T cells and antigen-pulsed B cells. Lastly, lamellipodia formation was investigated by assessing the ability of T cells to spread on anti-CD3 coated slides. Results Target genes downregulated by miR-181c were identified. One such target was BRK1, a component of the WAVE2 complex that has been shown to play a pivotal role in actin polymerisation. Validation experiments showed that overexpression and inhibition of miR-181c had no impact on BRK1 mRNA expression but did in fact modulate protein expression, suggesting that miR-181c regulates BRK1 at the translational level. We demonstrated that primary T cell activation resulted in downregulation of miR-181c and upregulation of BRK1 protein expression, further strengthening our hypothesis that the miR-181c-BRK1 axis may play an important role in T cell activation. Next, we found that loss of BRK1 resulted in reduced T cell activation as shown by decreased expression of CD69 and CD154. Furthermore, we showed that downregulation of BRK1 expression by shRNA resulted in reduced actin polymerisation after T cell stimulation. Reduced expression of BRK1 led to a marked reduction in the total area (in square micrometers) of F-actin accumulation at T cell contact sites and synapses with B cells indicating defective immune synapse formation. Moreover, reduced BRK1 expression resulted in defect in lamellipodia formation in response to T cell receptor stimulation. Similarly, ectopic expression of miR-181c in Jurkat T cells also led to a reduction in T cell activation and actin polymerisation coupled with defects in immune synapse and lamellipodia formation, hence confirming the important role of the miR-181c-BRK1 axis in T cell activation. Lastly, we demonstrated that suppression of BRK1 induced reduced expression of other pivotal proteins in the WAVE2 complex including WAVE2, Abi1 and Sra1. This suggests that impairment of actin polymerisation-dependent T cell functions were a result of instability of the WAVE2 complex following BRK1 suppression. Conclusion For the first time, we hereby demonstrate that BRK1 is a target of miR-181c. Moreover, we have highlighted the potential role of the miR-181c-BRK1 axis in impaired actin polymerisation-dependent T cell function and immune synapse formation. Deregulation of the miR-181c-BRK1 axis requires further evaluation in haematological malignancies. Disclosures No relevant conflicts of interest to declare.

Inhibitory Ligands CD200, CD270, CD274 and CD276 Are Expressed On Eμ-TCL1 Transgenic Mouse Splenocytes and Are of Potential Relevance to Impaired T-Cell Function in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 313-313
Author(s):  
Fabienne McClanahan ◽  
Cristina Ghirelli ◽  
Paul Greaves ◽  
John C. Riches ◽  
Rita Coutinho ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
Synapse Formation ◽  
Effector Function ◽  
Immune Synapse ◽  
Blocking Antibodies ◽  
Cell Effector Function

Abstract Abstract 313 Background: We have previously demonstrated that CD4 and CD8 T-cells from CLL patients show profound dysfunctions in multiple gene pathways, including the actin cytoskeleton, which impairs the formation of functional immunologic synapses between T cells and APCs. Functional screening assays on Mec-1 cells have identified CD200, CD270, CD274, and CD276 as inhibitory ligands which induce impaired actin synapse formation in both allogeneic and autologous T cells. We also demonstrated that the Eμ-TCL1 transgenic mouse model of CLL closely resembles the T-cell defects observed in humans, validating it as a valuable preclinical tool to examine changes in the microenvironment alongside the development of leukaemia. The aim of the current study is to investigate the role of CD200, CD270, CD274, and CD276 in the Eμ-TCL1 model. Methods: We used multiparameter flow cytometry to establish the expression of inhibitory ligands on CD19+/CD5+ unpurified splenocytes from Eμ-TCL1 mice on both the C57Bl/6 (B6) and the C3HB6-F1 background and compared this to unpurified splenocytes from age matched wild-type (WT) controls of the respective coisogenic strain. Results: A total of 19 leukemic Eμ-TCL1 (n=10 C57Bl/6 and n=9 C3HB6-F1 background) and 11 WT mice (n=6 C57Bl/6 and n=5 C3HB6-F1 background) were examined. CD19+/CD5+ CLL cells constituted 92% (range 62–97%) of the DAPI-negative lymphocyte population. On CD19+/CD5+ CLL cells, CD274 (mean 98% ± SEM 0.4) and CD200 (mean 84% ± SEM 2.9 were uniformly strongly expressed, while CD270 (mean 74% ± SEM 4.7) and CD276 (mean 50% ± SEM 6.6) showed a weaker and more diverse expression, with no significant differences between the two backgrounds (all p>.05). Similar expression patterns were observed in Eμ-TCL1 mice with spontaneously occurring CLL and transplanted transgenic mice, with no differences between spontaneous and induced CLL (all p>.05). We then compared transgenic CD19+/CD5+ CLL cells to the WT CD19+ and the WT CD19+/CD5+ B1a-like cell population. Eμ-TCL1 CLL splenocytes showed a significant higher expression of CD274 and CD276 compared to expression on WT CD19+ (p<.0001, p=.00349) splenocytes. When compared to WT B1a-like splenocytes, only CD274 was significantly higher expressed (p<.0001). To clarify the impact of genetic strain, B6 and C3HB6-F1 were investigated separately: transgenic mice on the B6 background showed significantly higher expression of CD274 compared to WT B6 CD19+ (p=.0015) and WT B6 B1a-like (p<.0001) splenocytes. In contrast, transgenic mice on the C3HB6-F1 background showed a significant higher expression of CD274 and CD276 compared to WT CD19+ (p=.0002, p=.00354) and WT B1a-like (p=.0005, p=.00384) splenocytes. These patterns substantiate differences of the expression of inhibitory ligands between the WT strains, but of note, these were not mirrored in TCL1 mice. In previous experiments, we used the Eμ-TCL1 model to investigate the polarization of F-actin and phosphotyrosine at the immune synapse between splenic autologous T-cells and APCs and subsequent effector function. Age-matched WT mice had a significantly higher accumulation than transgenic mice. To assess the functional role of inhibitory ligands, knock-down experiments using lentiviral shRNA and blocking antibodies are currently under way to assess if this restores immune synapse formation and T cell effector function in vivo. Conclusions: The inhibitory ligands CD200, CD270, CD274 and CD276 are expressed in vivo and appear to be of functional relevance for the anti-cancer immune response. They therefore represent attractive targets to restore T-cell effector function, which might be achieved by gene therapy approaches and blocking antibodies. Disclosures: Gribben: Celgene: Honoraria.

scholarly journals Retrograde And Anterograde Transport Of LAT-Vesicles During The Immunological Synapse Formation: Defining The Finely-Tuned Mechanism

2020 ◽  
Author(s):  
Juan José Saez ◽  
Stéphanie Dogniaux ◽  
Massiullah Shafaq-Zadah ◽  
Ludger Johannes ◽  
Claire Hivroz ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Synapse Formation ◽  
Immunological Synapse ◽  
Retrograde Transport ◽  
Anterograde Transport ◽  
Immune Synapse ◽  
Cellular Context ◽  
Intracellular Compartments

ABSTRACTLAT is an important player of the signaling cascade induced by TCR activation. This adapter molecule is present at the plasma membrane of T lymphocytes and more abundantly in intracellular compartments. Upon T-cell activation the intracellular pool of LAT is recruited to the immune synapse (IS). We previously described two pathways controlling LAT trafficking: retrograde transport from endosomes to the TGN, and anterograde traffic from the Golgi to the IS. We address the specific role of 4 proteins, the GTPase Rab6, the t-SNARE syntaxin-16, the v-SNARE VAMP7 and the golgin GMAP210, in each pathway. Using different methods (endocytosis and Golgi trap assays, confocal and TIRF microscopy, TCR-signalosome pull down) we show that syntaxin-16 is regulating the retrograde transport of LAT whereas VAMP7 is regulating the anterograde transport. Moreover, GMAP210 and Rab6, known to contribute in both pathways, are in our cellular context specifically and respectively involved in anterograde and retrograde transport of LAT. Altogether, our data describe how retrograde and anterograde pathways coordinate LAT enrichment at the IS and point the Golgi as a central hub for the polarized recruitment of LAT to the IS. The role that this finely-tuned transport of signaling molecules plays in T-cell activation is discussed.

scholarly journals Agent-Based Modeling of T Cell Receptor Cooperativity

2020 ◽  
Vol 21 (18) ◽  
pp. 6473
Author(s):  
Anastasios Siokis ◽  
Philippe A. Robert ◽  
Michael Meyer-Hermann
Keyword(s):  
T Cell ◽  
Synapse Formation ◽  
Immunological Synapse ◽  
Cell Receptor ◽  
Agent Based Model ◽  
Tcr Signaling ◽  
Immune Synapse ◽  
Agent Based ◽  
Affinity Modulation ◽  
Antigen Peptide

Immunological synapse (IS) formation is a key event during antigen recognition by T cells. Recent experimental evidence suggests that the affinity between T cell receptors (TCRs) and antigen is actively modulated during the early steps of TCR signaling. In this work, we used an agent-based model to study possible mechanisms for affinity modulation during IS formation. We show that, without any specific active mechanism, the observed affinity between receptors and ligands evolves over time and depends on the density of ligands of the antigen peptide presented by major histocompatibility complexes (pMHC) and TCR molecules. A comparison between the presence or absence of TCR–pMHC centrally directed flow due to F-actin coupling suggests that centripetal transport is a potential mechanism for affinity modulation. The model further suggests that the time point of affinity measurement during immune synapse formation is critical. Finally, a mathematical model of F-actin foci formation incorporated in the agent-based model shows that TCR affinity can potentially be actively modulated by positive/negative feedback of the F-actin foci on the TCR-pMHC association rate kon.

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