scholarly journals Immune synapse formation requires ZAP-70 recruitment by ezrin and CD43 removal by moesin

2007 ◽  
Vol 179 (4) ◽  
pp. 733-746 ◽  
Author(s):  
Tal Ilani ◽  
Chand Khanna ◽  
Ming Zhou ◽  
Timothy D. Veenstra ◽  
Anthony Bretscher
Keyword(s):  
T Cells ◽  
T Cell ◽  
Membrane Proteins ◽  
Intracellular Signaling ◽  
Synapse Formation ◽  
Immunological Synapse ◽  
Cell Cortex ◽  
Receptor Ligand ◽  
Signaling Molecule ◽  
Immune Synapse

Immunological synapse (IS) formation involves receptor–ligand pair clustering and intracellular signaling molecule recruitment with a coincident removal of other membrane proteins away from the IS. As microfilament–membrane linkage is critical to this process, we investigated the involvement of ezrin and moesin, the two ezrin/radixin/moesin proteins expressed in T cells. We demonstrate that ezrin and moesin, which are generally believed to be functionally redundant, are differentially localized and have important and complementary functions in IS formation. Specifically, we find that ezrin directly interacts with and recruits the signaling kinase ZAP-70 to the IS. Furthermore, the activation of ezrin by phosphorylation is essential for this process. In contrast, moesin dephosphorylation and removal, along with CD43, are necessary to prepare a region of the cell cortex for IS. Thus, ezrin and moesin have distinct and critical functions in the T cell cortex during IS formation.

Lenalidomide Repairs Suppressed T Cell Immunological Synapse Formation in Follicular Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 885-885 ◽  
Author(s):  
Alan G. Ramsay ◽  
Andrew J. Clear ◽  
Rewa Fatah ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Follicular Lymphoma ◽  
Cd8 T Cells ◽  
Molecular Basis ◽  
Synapse Formation ◽  
Immunological Synapse ◽  
Filamin A ◽  
Altered Expression ◽  
Immune Synapse

Abstract Identifying mechanisms of tumor-induced immune suppression will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired T cell responses in follicular lymphoma (FL) and demonstrate impaired T cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between tumor-infiltrating CD4 and CD8 T cells and autologous FL tumor cells with and without superantigen as antigen-presenting cells (APCs). We identified a significant reduction in formation of the T cell immune synapse in both CD4 (p<0.01) and CD8 T cells (p<0.01) from FL patients compared to age-matched healthy donors (> 50% reduction). Comparable immunological defects were also identified in CD4 and CD8 T cells from transformed-FL (t-FL) patients. This defect was induced by tumor contact since T cell defects were induced in healthy T cells when they were were co-cultured for 48 hr with either allogeneic FL or t-FL cells, but not with healthy allogeneic B cells (P < 0.01). Of interest there was enhanced suppression of CD4 T cell synapses following FL co-culture assays and CD8 T cells in the t-FL setting. Since previous gene expression profile studies (Dave et al. NEJM, 2003) demonstrated prognostic significance of altered expression of immune signature genes, we examined the molecular nature of the FL-induced T cell defect by quantifying recruitment of a number of these T cell cytoskeletal signaling proteins to the immune synapse. Following primary co-culture with FL cells, previously healthy T cells showed suppressed recruitment of integrin LFA-1, Lck, Itk, Rab27A and filamin-A to the synapse in subsequent T cell:APC interactions (P < 0.01). We further demonstrate that the immune synapse defects were repaired in part by treatment of the cells with the immunomodulatory drug lenalidomide. Of note, treatment of both FL B cells and autologous T cells with lenalidomide (1 μM for 24h) was required to enhance formation of the F-actin synapse and recruitment of tyrosine-phosphorylated protein and filamin-A irrespective of the presence of exogenous antigen (P < 0.01). We validated the altered expression of a number of these molecules including Itk, Rab27A and filamin-A at the protein level in FL tissue microarrays (TMA). Of note, elevated expression of intrafollicular Rab27A, that mediates targeted secretion of CD8 T cell cytolytic granules, was found in samples from a long-survival group (FL patients who lived more than 15 years). These results provide mechanistic insight into the demonstrated activity of lenalidomide in relapsed or refractory aggressive FL (Wiernik et al. JCO, 2008). These studies demonstrate the role of the tumor in the observed T cell defects in FL, and the molecular basis of these defects and suggest that immunotherapeutic approaches including the use lenalidomide offer the exciting prospect of repairing T cell suppression in B cell malignancies to enhance immune-mediated immunological responses against lymphoma cells.

Intermediate Filament (IF) Protein Vimentin Regulates T Cell Mediated Immune Response in Gvhd

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3073-3073 ◽  
Author(s):  
Dan Li ◽  
Patenia Rebecca ◽  
Miguel A. Cruz ◽  
Jeffrey J. Molldrem ◽  
Richard E. Champlin ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Adhesion ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Synapse Formation ◽  
Immunological Synapse ◽  
Immune Synapse ◽  
T Cell Adhesion

Abstract Graft-versus-host-disease (GVHD) is an alloimmune response complicating allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although donor T lymphocytes and recipient antigen presenting cells (APCs) are the primarily mediators of GVHD, the molecular and cellular basis are not well understood. Recent published studies investigated T cell migration and homing in GVHD, especially effects of adhesion molecules including chemokines and integrins. Intermediate filaments (IFs) are cytoskeletal polymers encoded by a large family of differentially expressed genes that provide crucial structural support. As the most abundant IF protein and the only known IF protein in leukocytes, vimentin plays an important role in stabilizing intracellular architecture, maintaining cellular integrity and providing resistance against stress. Vimentin-deficient (Vim-/-) mice are viable with impaired wound healing, and defects in PMN and lymphocyte adhesion to endothelial cells. In addition, Vimentin has been reported to be involved in colitis, Crohn's disease and allograft rejection. Herein, we report that vimentin regulates the organization of proteins in cell adhesion, migration and signaling, all of which are important for T cell activation in GVHD. Using homotypic aggregation assay, we found there was significantly reduced aggregation of primary T cells isolated from Vim-/- mice compared to WT control. However, the expression of both LFA-1 and ICAM-1 are similar between WT and Vim-/- T cells, thus the reduced T cell adhesion is not attributed to LFA-1 expression. We further investigated whether vimentin regulates mouse primary T cell proliferation upon activation in mixed lymphocyte reactions (MLR), and demonstrated there was a significant reduction of both CD4+ and CD8+ T cell proliferation in the absence of vimentin (Vim-/-) compared to WT control. Moreover, the frequency of IFN-γ (Th1) and IL-17 (Th17) producing CD4+ cells was significantly reduced in T cells isolated from Vim-/- mice compared to WT control. To investigate the role of vimentin in regulating TCR-induced activation, we examined the immune synapse formation in mouse CD8+ T cells, and found vimentin was enriched proximal to the membrane and associated with the prominent CD3/LFA-1 cluster upon TCR stimulation. In the control without any TCR stimulation, the predominant pattern was different with vimentin evenly distributed beneath the cell membrane. In the absence of vimentin (Vim-/-), immune synapse failed to form in mouse CD8+ T cells upon TCR stimulation. The data demonstrate that vimentin can regulate immunological synapse function and signal transduction in T cell activation and proliferation. To determine whether vimentin expression in T cells plays a role in GVHD, the MHC class I and II disparate model, C57BL/6 (H-2b) to BALB/c (H-2d) transplantation, was used to establish GVHD. T cells from WT or Vim-/- (C57BL/6 background) mice were used as donors and Balb/c mice as recipients. Irradiated BALB/c mice received 5x106 T cell-depleted bone marrow cells (WT) and 10×106 T cells (WT or Vim-/-). In comparison to WT control, mice received Vim-/- T cells showed a lower mortality rate. Within 8 weeks post-transplantation, about 65% of mice received Vim-/- T cells survived, compared with only less than 10% of control mice received WT T cells (P=0.036; n=15 in each group). Control recipients had severe GVHD in the skin, intestine and liver. Mice received Vim-/- T cells exhibited only mild changes in these organs, reflected in their significantly lower GVHD scores. There were significantly reduced donor-derived CD4+ and CD8+ T cells in secondary lymphoid organs. Thus, the reduced homing and proliferation of Vim-/- T cells in vivo led to the reduced mortality and morbidity associated with GVHD. In summary, we have shown that there are significantly reduced T cell adhesion, proliferation and Th1/17 polarization in Vim-/- T cells, and vimentin participates in TCR clustering and immunological synapse formation in CD8+ T cells. Furthermore, vimentin plays an important role in GVHD through regulating donor T cell adhesion, proliferation and activation. Our data will not only significantly advance our knowledge of GVHD, but also define a new function of vimentin and IF proteins in health and diseases, and thus provide a rationale for using vimentin inhibitors as potential novel therapeutic interventions for GVHD. Disclosures No relevant conflicts of interest to declare.

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.

scholarly journals Periodic Membrane Potential and Ca2+ Oscillations in T Cells Forming an Immune Synapse

2020 ◽  
Vol 21 (5) ◽  
pp. 1568 ◽  
Author(s):  
Ferenc Papp ◽  
Peter Hajdu ◽  
Gabor Tajti ◽  
Agnes Toth ◽  
Eva Nagy ◽  
...  
Keyword(s):  
T Cells ◽  
Membrane Potential ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Calcium Release ◽  
Immunological Synapse ◽  
Level Control ◽  
Potential Oscillations ◽  
Immune Synapse

The immunological synapse (IS) is a specialized contact area formed between a T cell and an antigen presenting cell (APC). Besides molecules directly involved in antigen recognition such as the TCR/CD3 complex, ion channels important in the membrane potential and intracellular free Ca2+ concentration control of T cells are also recruited into the IS. These are the voltage-gated Kv1.3 and Ca2+-activated KCa3.1 K+ channels and the calcium release-activated Ca2+ channel (CRAC). However, the consequence of this recruitment on membrane potential and Ca2+ level control is not known. Here we demonstrate that the membrane potential (MP) of murine T cells conjugated with APCs in an IS shows characteristic oscillations. We found that depolarization of the membrane by current injection or by increased extracellular K+ concentration produced membrane potential oscillations (MPO) significantly more frequently in conjugated T cells than in lone T cells. Furthermore, oscillation of the free intracellular Ca2+ concentration could also be observed more frequently in cells forming an IS than in lone cells. We suggest that in the IS the special arrangement of channels and the constrained space between the interacting cells creates a favorable environment for these oscillations, which may enhance the signaling process leading to T cell activation.

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.

CC-122 Repairs T Cell Activation in Chronic Lymphocytic Leukemia That Results in a Concomitant Increase in PD-1:PD-L1 and CTLA-4 Immune Checkpoint Expression at the Immunological Synapse

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1738-1738
Author(s):  
Benedetta Apollonio ◽  
Mariam Fanous ◽  
Mohamed-Reda Benmebarek ◽  
Stephen Devereux ◽  
Patrick Hagner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Immunological Synapse ◽  
Lymphocytic Leukemia ◽  
Immune Synapse ◽  
Cell Synapse

Abstract Immunomodulatory drugs (IMiDs®) such as lenalidomide and immune checkpoint blockade (ICB) antibodies can enhance autologous anti-tumor T cell immunity and have the potential to elicit durable control of disease in B cell malignancies. These immunotherapies are likely to be most effective when employed in treatment combinations. Thus, the goal of pre-clinical research should be to reveal mechanisms of action (MOA) in the tumor microenvironment (TME) and identify biomarkers to guide development of combination therapy for patients. CC-122 is a novel first-in-class pleiotropic pathway modifier (PPM®) that has potent anti-proliferative, anti-angiogenic and immunomodulatory activities and is currently in Phase I clinical trials for lymphoma and chronic lymphocytic leukemia (CLL). Here, we have utilized the immunological synapse bioassay to examine T cell interactions with CLL tumor cells (modeling anti-tumor T cell responses in the TME) following CC-122 treatment and measure the expression of co-signaling complexes at the synapse. Conjugation assays and confocal imaging were used to visualize intercellular conjugate interactions and F-actin polymerization at the immune synapse between CD4+ and CD8+ T cells and autologous CLL tumor cells pulsed with superantigen (acting as antigen-presenting cells, APCs). Peripheral blood was obtained from treatment naive CLL patients (n=40) representative of disease heterogeneity. Treatment of both purified CLL cells and CD4+ or CD8+ T cells with CC-122 (0.01 - 1 μM for 24h) dramatically enhanced the number of T cells recognizing tumor cells (% conjugation) and increased the formation of F-actin immune synapses (area, μm2) compared to vehicle treated cells (P<.01). Notably, CC-122 treatment induced T cells to engage in multiple tumor cell synapse interactions that were more pronounced in restored CD8+ T cell lytic synapses. This immunomodulatory activity was detected across all CLL patient samples and drug concentrations tested. In addition, synapse strength as measured by total fluorescence intensity of F-actin per T cell:APC conjugate increased significantly with CC-122 (P<.01). A critical MOA of lenalidomide is activation of T cell immune synapse signaling. Here, our comparative studies revealed that CC-122 (0.1 - 1 μM) significantly enhanced autologous T cell synapse activity in CLL by 4 - 5 fold versus vehicle (P<.01), whereas lenalidomide (1 μM) enhanced activity by 3 fold vs vehicle. Moreover, CC-122 treatment resulted in increased expression and polarization of tyrosine-phosphorylated proteins at T cell synapses compared to lenalidomide and vehicle treatment (P<.01). This data provides evidence that CC-122 induces functional T cell synapses that control the assembly of signaling complexes between the T cell receptor (TCR) and the F-actin cytoskeletal layer. Following T cell recognition of APCs, co-signaling receptors co-localize at the immune synapse where they synergize with TCR signaling to promote (co-stimulatory receptors) or inhibit (co-inhibitory/'immune checkpoint' receptors) T cell activation. Quantitative image analysis studies revealed that restoration of T cell synapse activity with CC-122 was accompanied by an increased recruitment of inducible co-stimulator (ICOS) to the synapse that was dose-dependent (P<.01). CC-122 treatment also increased polarized expression of CTLA-4 and PD-1 immune checkpoint proteins at the synapse with PD-L1+ tumor cells. The observed up-regulation of co-inhibitory receptors led to combining CC-122 with anti-PD-L1, anti-PD-1 or anti-CTLA-4 blocking antibodies. Results show that these treatment combinations increased T cell synapse activity compared to using these immunotherapies alone (P<.01). In conclusion, our results demonstrate for the first time that CC-122 can activate T cell immune synapse signaling against autologous CLL tumor cells and this immunomodulatory capability is more potent than lenalidomide. We further show that CC-122 activation of T cells is associated with enhanced expression of the co-stimulatory receptor ICOS and co-inhibitory checkpoints CTLA-4 and PD-1 at the synapse site. Importantly, our pre-clinical data demonstrates that this regulatory feedback inhibition can be exploited by the addition of anti-PD-L1, anti-PD-1 or anti-CTLA-4 ICB to CC-122 to more optimally stimulate T cell activity against immunosuppressive tumor cells. Disclosures Hagner: Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment. Gandhi:Celgene: Employment, Equity Ownership. Ramsay:MedImmune: Research Funding; Celgene: Research Funding.

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