Cdc42-mediated MTOC polarization in dendritic cells controls targeted delivery of cytokines at the immune synapse

The immune synapse (IS) forms as dendritic cells (DCs) and T cells interact in lymph nodes during initiation of adaptive immunity. Factors that contribute to the formation and maintenance of IS stability and function have been mostly studied in T cells, whereas little is known about events occurring during synapse formation in DCs. Here, we show that DCs activated by Toll-like receptor (TLR) agonists reorient the microtubule-organizing center (MTOC) toward the interacting T cell during antigen-specific synapse formation through a mechanism that depends on the Rho GTPase Cdc42. IL-12, a pivotal cytokine produced by DCs, is found enriched around the MTOC at early time points after TLR ligation and is dragged to the DC–T cell interface in antigen-specific synapses. Synaptic delivery of IL-12 induces activation of pSTAT4 and IFN-γ neosynthesis in CD8+ naive T cells engaged in antigen-specific conjugates and promotes the survival of antigen-primed T cells. We propose that DC polarization increases the local concentration of proinflammatory mediators at the IS and that this represents a new mechanism by which T cell priming is controlled.

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Cytoskeletal remodeling mediated by WASp in dendritic cells is necessary for normal immune synapse formation and T-cell priming

Blood ◽

10.1182/blood-2011-03-340265 ◽

2011 ◽

Vol 118 (9) ◽

pp. 2492-2501 ◽

Cited By ~ 54

Author(s):

Gerben Bouma ◽

Ariadna Mendoza-Naranjo ◽

Michael P. Blundell ◽

Elena de Falco ◽

Kathryn L. Parsley ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Synapse Formation ◽

Critical Role ◽

Calcium Flux ◽

Microtubule Organizing Center ◽

Specific System ◽

Hematopoietic Stem ◽

T Cell Priming

Abstract Rearrangement of the cytoskeleton in T cells plays a critical role in the organization of a complex signaling interface referred to as immunologic synapse (IS). Surprisingly, the contribution of antigen presenting cells, in particular dendritic cells (DCs), to the structure and function of the IS has not been investigated in as much detail. We have used a natural model of cytoskeletal dysfunction caused by deficiency of the Wiskott-Aldrich syndrome protein (WASp) to explore the contribution of the DC cytoskeleton to IS formation and to T-cell priming. In an antigen-specific system, T-DC contacts were found to be less stable when DCs alone lacked WASp, and associated with multiple defects of IS structure. As a consequence, DCs were unable to support normal IL-12 secretion, and events downstream of TCR signaling were abrogated, including increased calcium flux, microtubule organizing center (MTOC) polarization, phosphorylation of ZAP-70, and T-cell proliferation. Formation of an effective signaling interface is therefore dependent on active cytoskeletal rearrangements in DCs even when T cells are functionally competent. Deficiency of DC-mediated activities may contribute significantly to the varied immunodysregulation observed in patients with WAS, and also in those with limited myeloid reconstitution after allogeneic hematopoietic stem cell transplantation.

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A novel form of immune signaling revealed by transmission of the inflammatory mediator serotonin between dendritic cells and T cells

Blood ◽

10.1182/blood-2005-07-2903 ◽

2006 ◽

Vol 107 (3) ◽

pp. 1010-1017 ◽

Cited By ~ 138

Author(s):

Peta J. O'Connell ◽

Xiangbin Wang ◽

Matilde Leon-Ponte ◽

Corrie Griffiths ◽

Sandeep C. Pingle ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Activated T Cells ◽

Immune Synapse ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation ◽

5 Ht Uptake

AbstractAdaptive immunity is triggered at the immune synapse, where peptide-major histocompatibility complexes and costimulatory molecules expressed by dendritic cells (DCs) are physically presented to T cells. Here we describe transmission of the inflammatory monoamine serotonin (5-hydroxytryptamine [5-HT]) between these cells. DCs take up 5-HT from the microenvironment and from activated T cells (that synthesize 5-HT) and this uptake is inhibited by the antidepressant, fluoxetine. Expression of 5-HT transporters (SERTs) is regulated by DC maturation, exposure to microbial stimuli, and physical interactions with T cells. Significantly, 5-HT sequestered by DCs is stored within LAMP-1+ vesicles and subsequently released via Ca2+-dependent exocytosis, which was confirmed by amperometric recordings. In turn, extracellular 5-HT can reduce T-cell levels of cAMP, a modulator of T-cell activation. Thus, through the uptake of 5-HT at sites of inflammation, and from activated T cells, DCs may shuttle 5-HT to naive T cells and thereby modulate T-cell proliferation and differentiation. These data constitute the first direct measurement of triggered exocytosis by DCs and reveal a new and rapid type of signaling that may be optimized by the intimate synaptic environment between DCs and T cells. Moreover, these results highlight an important role for 5-HT signaling in immune function and the potential consequences of commonly used drugs that target 5-HT uptake and release.

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Imaging of plasmacytoid dendritic cell interactions with T cells

Blood ◽

10.1182/blood-2008-02-139865 ◽

2009 ◽

Vol 113 (1) ◽

pp. 75-84 ◽

Cited By ~ 33

Author(s):

María Mittelbrunn ◽

Gloria Martínez del Hoyo ◽

María López-Bravo ◽

Noa B. Martín-Cofreces ◽

Alix Scholer ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Plasmacytoid Dendritic Cell ◽

Time Lapse ◽

Cell Interactions ◽

Type I ◽

Microtubule Organizing Center ◽

Immune Synapse ◽

Organizing Center

Abstract Plasmacytoid dendritic cells (pDCs) efficiently produce type I interferon and participate in adaptive immune responses, although the molecular interactions between pDCs and antigen-specific T cells remain unknown. This study examines immune synapse (IS) formation between murine pDCs and CD4+ T cells. Mature pDCs formed canonical ISs, involving relocation to the contact site of the microtubule-organizing center, F-actin, protein kinase C-θ, and pVav, and activation of early signaling molecules in T cells. However, immature pDCs were less efficient at forming conjugates with T cells and inducing IS formation, microtubule-organizing center translocation, and T-cell signaling and activation. Time-lapse videomicroscopy and 2-photon in vivo imaging of pDC–T-cell interactions revealed that immature pDCs preferentially mediated transient interactions, whereas mature pDCs promoted more stable contacts. Our data indicate that, under steady-state conditions, pDCs preferentially establish transient contacts with naive T cells and show a very modest immunogenic capability, whereas on maturation, pDCs are able to form long-lived contacts with T cells and significantly enhance their capacity to activate these lymphocytes.

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DC-SIGN–mediated Infectious Synapse Formation Enhances X4 HIV-1 Transmission from Dendritic Cells to T Cells

Journal of Experimental Medicine ◽

10.1084/jem.20041356 ◽

2004 ◽

Vol 200 (10) ◽

pp. 1279-1288 ◽

Cited By ~ 182

Author(s):

Jean-François Arrighi ◽

Marjorie Pion ◽

Eduardo Garcia ◽

Jean-Michel Escola ◽

Yvette van Kooyk ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Hiv Infection ◽

Cd4 T Cells ◽

Synapse Formation ◽

Model Systems ◽

Hiv 1 ◽

Infectious Synapse

Dendritic cells (DCs) are essential for the early events of human immunodeficiency virus (HIV) infection. Model systems of HIV sexual transmission have shown that DCs expressing the DC-specific C-type lectin DC-SIGN capture and internalize HIV at mucosal surfaces and efficiently transfer HIV to CD4+ T cells in lymph nodes, where viral replication occurs. Upon DC–T cell clustering, internalized HIV accumulates on the DC side at the contact zone (infectious synapse), between DCs and T cells, whereas HIV receptors and coreceptors are enriched on the T cell side. Viral concentration at the infectious synapse may explain, at least in part, why DC transmission of HIV to T cells is so efficient. Here, we have investigated the role of DC-SIGN on primary DCs in X4 HIV-1 capture and transmission using small interfering RNA–expressing lentiviral vectors to specifically knockdown DC-SIGN. We demonstrate that DC-SIGN− DCs internalize X4 HIV-1 as well as DC-SIGN+ DCs, although binding of virions is reduced. Strikingly, DC-SIGN knockdown in DCs selectively impairs infectious synapse formation between DCs and resting CD4+ T cells, but does not prevent the formation of DC–T cells conjugates. Our results demonstrate that DC-SIGN is required downstream from viral capture for the formation of the infectious synapse between DCs and T cells. These findings provide a novel explanation for the role of DC-SIGN in the transfer and enhancement of HIV infection from DCs to T cells, a crucial step for HIV transmission and pathogenesis.

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

Blood ◽

10.1182/blood.v116.21.696.696 ◽

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.

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Stimulation of CD8+ T Cells following Diphtheria Toxin-Mediated Antigen Delivery into Dendritic Cells

Infection and Immunity ◽

10.1128/iai.74.2.1001-1008.2006 ◽

2006 ◽

Vol 74 (2) ◽

pp. 1001-1008 ◽

Cited By ~ 10

Author(s):

Christine A. Shaw ◽

Michael N. Starnbach

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Targeted Delivery ◽

Diphtheria Toxin ◽

Mammalian Cells ◽

T Cell Response ◽

Host Cells ◽

Target Antigen ◽

Antigen Delivery

ABSTRACT Recognition and clearance of many intracellular pathogens requires the activation and subsequent effector functions of CD8+ T lymphocytes. To stimulate CD8+ T cells by immunization, the target antigens must be delivered into the cytosol of host cells. There they can be processed into peptides and presented in the context of major histocompatibility complex class I molecules to antigen-specific CD8+ T cells. One method of delivering antigens into the cytosol is to fuse them to modified bacterial toxins that are able to enter mammalian cells. The expression pattern of the toxin receptors in the host will determine the cell population that the toxin fusion protein targets and will thus restrict antigen-specific T-cell recognition to the same population. In this study we describe the development and characterization of a diphtheria toxin (DT)-based antigen delivery system. Using CD11c-DTR transgenic mice that express the DT receptor in dendritic cells (DC), this system allows for targeted delivery of CD8+ T-cell antigen to DC. We show that antigen-specific CD8+ T cells proliferate in CD11c-DTR mice following immunization with catalytically inactive DT-antigen fusion proteins. We also show that a toxin-based system that restricts antigen delivery to DC results in more robust antigen-specific CD8+ T-cell proliferation than a toxin-based system that does not restrict delivery to a particular cell type. These results have implications for vaccine design, and they suggest that use of a toxin-based vector to target antigen to DC may be an effective way to induce a CD8+ T-cell response.

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

Blood ◽

10.1182/blood-2021-146488 ◽

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 > huCD33 + huCD86 + PD-L1 + Ba/F3: 7,749 > 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<0.0001] vs 7.7 % [p<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.

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Follicular lymphoma cells induce T-cell immunologic synapse dysfunction that can be repaired with lenalidomide: implications for the tumor microenvironment and immunotherapy

Blood ◽

10.1182/blood-2009-04-217687 ◽

2009 ◽

Vol 114 (21) ◽

pp. 4713-4720 ◽

Cited By ~ 150

Author(s):

Alan G. Ramsay ◽

Andrew J. Clear ◽

Gavin Kelly ◽

Rewas Fatah ◽

Janet Matthews ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Follicular Lymphoma ◽

Cancer Progression ◽

Synapse Formation ◽

B Cell Lymphoma ◽

Cell Contact ◽

Immune Recognition ◽

Immune Synapse ◽

Immunologic Synapse

Abstract An important hallmark of cancer progression is the ability of tumor cells to evade immune recognition. Understanding the relationship between neoplastic cells and the immune microenvironment should facilitate the design of improved immunotherapies. Here we identify impaired T-cell immunologic synapse formation as an active immunosuppressive mechanism in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). We found a significant reduction in formation of the F-actin immune synapse in tumor-infiltrating T cells (P < .01) from lymphoma patients compared with age-matched healthy donor cells. Peripheral blood T cells exhibited this defect only in patients with leukemic-phase disease. Moreover, we demonstrate that this T-cell defect is induced after short-term tumor cell contact. After 24-hour coculture with FL cells, previously healthy T cells showed suppressed recruitment of critical signaling proteins to the synapse. We further demonstrate repair of this defect after treatment of both FL cells and T cells with the immunomodulatory drug lenalidomide. Tissue microarray analysis identified reduced expression of the T-cell synapse signature proteins, including the cytolytic effector molecule Rab27A associated with poor prognosis, in addition to reduced T-cell numbers and activity with disease transformation. Our results highlight the importance of identifying biomarkers and immunotherapeutic treatments for repairing T-cell responses in lymphoma.

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Leveraging a physiologically based quantitative translational modeling platform for designing bispecific T cell engagers for treatment of multiple myeloma

10.1101/2021.12.06.471352 ◽

2021 ◽

Author(s):

Tomoki Yoneyama ◽

Mi-Sook Kim ◽

Konstantin Piatkov ◽

Haiqing Wang ◽

Andy Z.X. Zhu

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Tumor Cell ◽

Tumor Cells ◽

Synapse Formation ◽

Modeling Framework ◽

Immune Synapse ◽

Immune Synapses ◽

Physiologically Based

Bispecific T cell engager (TCE) is an emerging anti-cancer modality which redirects cytotoxic T cells to tumor cells expressing tumor-associated antigen (TAA) thereby forming immune synapses to exerts anti-tumor effects. Considering the protein engineering challenges in designing and optimizing size and pharmacokinetically acceptable TCEs in the context of the complexity of intercellular bridging between T cells and tumor cells, a physiologically relevant and clinically verified computational modeling framework is of crucial importance to guide the process to understand the protein engineering trade offs. In this study, we developed a quantitative, physiologically based computational framework to predict immune synapse formation for a variety of molecular format of TCEs in tumor tissue. Our model incorporated the molecular size dependent biodistribution using the two pore theory, extra-vascularization of T cells and hematologic cancer cells, mechanistic bispecific intercellular binding of TCEs and competitive inhibitory interaction by shed targets. The biodistribution of TCE was verified by positron emission tomography imaging of [89Zr]AMG211 (a carcinoembryonic antigen-targeting TCE) in patients. Parameter sensitivity analyses indicated that immune synapse formation was highly sensitive to TAA expression, degree of target shedding and binding selectivity to tumor cell surface TAA over shed target. Interestingly, the model suggested a “sweet spot” for TCE’s CD3 binding affinity which balanced the trapping of TCE in T cell rich organs. The final model simulations indicated that the number of immune synapses is similar (~50/tumor cell) between two distinct clinical stage B cell maturation antigen (BCMA)-targeting TCEs, PF-06863135 in IgG format and AMG420 in BiTE format, at their respective efficacious dose in multiple myeloma patients, demonstrating the applicability of the developed computational modeling framework to molecular design optimization and clinical benchmarking for TCEs. This framework can be employed to other targets to provide a quantitative means to facilitate the model-informed best in class TCE discovery and development.

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Lenalidomide Repairs Suppressed T Cell Immunological Synapse Formation in Follicular Lymphoma

Blood ◽

10.1182/blood.v112.11.885.885 ◽

2008 ◽

Vol 112 (11) ◽

pp. 885-885 ◽

Cited By ~ 1

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.

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