Immunological Synapse Formation Research Articles

Venetoclax acts as an immunometabolic modulator to potentiate adoptive NK cell immunotherapy against leukemia.

Natural killer (NK) cell-based immunotherapy holds promise for cancer treatment; however, its efficacy remains limited, necessitating the development of alternative strategies. Here, we report that venetoclax, an FDA-approved BCL-2 inhibitor, directly activates NK cells, enhancing their cytotoxicity against acute myeloid leukemia (AML) both invitro and invivo, likely independent of BCL-2 inhibition. Through comprehensive approaches, including bulk and single-cell RNA sequencing, avidity measurement, and functional assays, we demonstrate that venetoclax increases the avidity of NK cells to AML cells and promotes lytic granule polarization during immunological synapse (IS) formation. Notably, we identify a distinct CD161lowCD218b+ NK cellsubpopulation that exhibits remarkable sensitivity to venetoclax treatment. Furthermore, venetoclax promotes mitochondrial respiration and ATP synthesis via the NF-κB pathway, thereby facilitating IS formation in NK cells. Collectively, our findings establish venetoclax as a multifaceted immunometabolic modulator of NK cell function and provide a promising strategy for augmenting NK cell-based cancer immunotherapy.

Individualized Multimodal Immunotherapy (IMI): Scientific Rationale and Clinical Experience from a Single Institution.

Oncolytic viruses and combinatorial immunotherapy for cancer (this Special Issue) are both part of cancer treatment at IOZK. This review focusses on an individual multimodal cancer immunotherapy concept developed by IOZK, Cologne, Germany. The scientific rationale for employing three main components is explained: (i) oncolytic Newcastle disease virus, (ii) modulated electrohyperthermia and (iii) individual tumor antigen and oncolytic virus modified dendritic cell vaccine (IO-VACR). The strategy involves repeated cancer-immunity cycles evoked in cancer patients by systemic oncolytic virus exposure plus hyperthermia pretreatment to induce immunogenic cell death followed by intradermal IO-VACR vaccination. As an example of the experience at IOZK, we present the latest results from combining the immunotherapy with standard treatment of patients suffering from glioblastoma multiforme. The promising clinical results in terms of overall survival benefit of additional individualized multimodal immunotherapy are presented. The cancer-immunity cycle, as introduced 10 years ago, describes key important steps occurring locally at the sites of both tumor and draining lymph nodes. This view is extended here towards systemic events occuring in blood where immunogenic cell death-induced tumor antigens are transported into the bone marrow. For 20 years it has been known that bone marrow is an antigen-responsive organ in which dendritic cells present tumor antigens to T cells leading to immunological synapse formation, tumor antigen-specific T cell activation and memory T cell formation. Bone marrow is known to be the most prominent source of de novo cellular generation in the body and to play an important role for the storage and maintenance of immunological memory. Its systemic activation is recommended to augment cancer-immunity cycles.

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Ex Vivo Enriched Tumor Antigen-Specific T Cells Are Optimal Effectors of T Cell Engagers and Tumor-Specific T Cell Receptor/T Cell Engager Combination Provides Synergistic Efficacy Against Hematologic Malignancies

Multiple myeloma (MM) and acute myeloid leukemia (AML) are blood cancers that remain difficult to treat because a large proportion of patients have disease that does not respond to currently available therapies. NexImmune's Artificial Immune Modulation (AIM) platform mimics natural dendritic cell function by using nanoparticles engineered with MHC molecules loaded with tumor specific peptides (signal 1) and anti-CD28 (signal 2) for activation and expansion of HLA A*02:01 restricted tumor associated antigen (TAA)-specific cytotoxic CD8 T lymphocytes (CTL). Additionally, T cell engager (TCE) therapy allows for TAA-specific, but T cell receptor (TCR) independent T cell cytotoxicity mediated by immunological synapse formation between targeted cancer cells and T cells. Given that T cell function in MM and AML patients is often perturbed and the response to TCE therapy is determined by pre-existing endogenous T cells of the patient, we hypothesized adoptively transferred multi-antigen specific T cells with a rejuvenated phenotype in combination with TCE therapy might produce optimal efficacy. We compared polyclonal CTL with AIM CTL (CTL that were AIM primed and expanded for optimal antigen experience and specificity). We evaluated the preclinical efficacy of B cell maturation antigen (BCMA)xCD3 TCE+MM-specific AIM CTL combination therapy and CD123xCD3 TCE+AML-specific AIM CTL combination therapy both in vitro and in vivo. In co-culture assays with polyclonal CTL and AIM CTL, we investigated the effect of prior antigen exposure on the efficacy of the TCE against tumor cell lines by measuring the responses to T cells alone and in the presence of TCE treatment. We quantified tumor cell survival and thus determined cytotoxicity of the various culture conditions by luciferase assay. Our analysis showed that, as expected, CD4 T cells had little potency. Non-HLA matched control CTL (bulk or enriched for naïve cells) without TCE showed higher cytotoxicity (data not shown), but these cells were much less potent than AIM CTL (data not shown and Fig. 1A). Combined TCR and TCE-mediated target cell killing was most efficient when using AIM CTL as effector cells (Fig. 1B). Correcting the data for TCR mediated killing revealed that antigen-experienced AIM CTL were indeed superior effectors of TCE potency than antigen inexperienced control CTL. This finding was reproducible regardless of the HLA type of the target cells, consistent with the TCR independent mechanism of action of TCE-mediated killing. Nuclear factor of activated T cells (NFAT) transcriptional reporter assays with Jurkat effector cells and AML target cells revealed that TCE treatment decreased NFAT transcriptional activity in TCE effector cells; moreover, TCE treatment in vivo (treating mice with AIM CTL +/- TCE) increased CD3 levels on AIM CTL. Both observations are consistent with reduced TCR mediated T cell activation in the context of TCE therapy. Furthermore, BCMAxCD3 TCE treatment of mice in a MM xenograft model resulted in decreased PD-1 expression on AIM CTL and enhanced the in vivo efficacy of AIM CTL. Our studies show that AIM CTL display strong anti-tumor activity, and even subtherapeutic AIM CTL cell doses can be highly efficacious when armed with a TCE. Importantly, our data indicate that AIM CTL cells are optimal TCE effectors, superior to less experienced, polyclonal CTL (such as the patient's endogenous T cells). Combining AIM CTL with TCE therapy has the additional benefits of complementary targets and mechanisms of action, reducing the risk of immune escape. Moreover, our preclinical studies suggest that TCE treatment reduces NFAT transcriptional activity and PD-1 induction associated with physiological TCR mediated T cell activation. This suggests that TCE mediated T cell activation can partially suppress TCR mediated T cell activation in AIM CTL, resulting in excellent tumor-specific cytolytic activity while inducing a transcriptional signature that preserves T cell fitness. Following withdrawal of TCE therapy, less exhausted AIM CTL will be present, facilitating the persistence of long-lasting anti-tumor activity and immunosurveillance. Combination of AIM CTL (autologous or HLA matched allogeneic) with TCE therapy is therefore a highly promising and innovative strategy for the induction of durable antitumor activity, and this approach has potential for clinical translation as a novel combinatorial immunotherapy.

Conformation-selective rather than avidity-based binding to tumor associated antigen derived peptide-MHC enables targeting of WT1-pMHC low expressing cancer cells by anti-WT1-pMHC/CD3 T cell engagers.

T cell engagers, a category of T cell-retargeting immunotherapy, are rapidly transforming clinical cancer care. However, the lack of tumor-specific targets poses a significant roadblock for broad adaptation of this therapeutic modality in many indications, often resulting in systemic on-target off-tumor toxicity. Though various tumor-derived intracellular mutations provide a massive pool of potential tumor-specific antigens, targeting them is extremely challenging, partly due to the low copy number of tumor associated antigen (TAA)-derived pMHC on tumor cell surface. Further, the interplay of binding geometry and format valency in relation to the capacity of a T cell engager to efficiently target low density cell-surface pMHC is not well understood. Using the Wilms' tumor 1 (WT1) oncoprotein as a proof-of-principle TAA, combined with an array of IgG-like T cell engager modalities that differ in their anti-TAA valency and binding geometry, we show that the ability to induce an immunological synapse formation, resulting in potent killing of WT1 positive cancer cell lines is primarily dependent on the distinct geometrical conformations between the Fab arms of anti-WT1-HLA-A*02:01 and anti-CD3. The augmented avidity conferred by the binding of two anti-WT1-HLA-A*02:01 Fab arms has only minimal influence on cell killing potency. These findings demonstrate the need for careful examination of key design parameters for the development of next-generation T cell engagers targeting low density TAA-pMHCs on tumor cells.

Integrin α3 promotes TH17 cell polarization and extravasation during autoimmune neuroinflammation.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by CNS-infiltrating leukocytes, including TH17 cells that are critical mediators of disease pathogenesis. Although targeting leukocyte trafficking is effective in treating autoimmunity, there are currently no therapeutic interventions that specifically block encephalitogenic TH17 cell migration. Here, we report integrin α3 as a TH17 cell-selective determinant of pathogenicity in experimental autoimmune encephalomyelitis. CNS-infiltrating TH17 cells express high integrin α3, and its deletion in CD4+ T cells or Il17a fate-mapped cells attenuated disease severity. Mechanistically, integrin α3 enhanced the immunological synapse formation to promote the polarization and proliferation of TH17 cells. Moreover, the transmigration of TH17 cells into the CNS was dependent on integrin α3, and integrin α3 deficiency enhanced the retention of CD4+ T cells in the perivascular space of the blood-brain barrier. Integrin α3-dependent interactions continuously maintain TH17 cell identity and effector function. The requirement of integrin α3 in TH17 cell pathogenicity suggests integrin α3 as a therapeutic target for MS treatment.

Crosstalk within peripheral blood mononuclear cells mediates anti-inflammatory effects of n-3 PUFA-rich lipid emulsions in parenteral nutrition

Parenteral nutrition (PN) rich in n-6 and n-3 long-chain fatty acids is used in clinical practice for nourishing patients who are unable to receive adequate nutrition through their digestive systems. In this study, we compare the effect on inflammation of the commonly used lipid emulsions Omegaven (n-3-rich) and Intralipid (n-6-rich) in human peripheral blood mononuclear cells (PBMCs). PBMCs were treated with different doses of n-3-rich Omegaven and n-6-rich Intralipid and the immune cells were characterized by flow cytometry. We show that incubation of PBMCs with n-3-rich Omegaven leads to an increase in expression of CD1d and CD86 in CD14+monocytes. At the same time, an increased number of NKT cells expressing cytotoxic T cell antigen 4 is observed, suggesting immunological synapse formation. Both CD14+monocytes and NKT cells showed an increase in IL-10 production and a reduction in the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-4, which led to an increase in the number of FOXP3+T regulatory cells. In addition, we show that n-3-rich Omegaven reduces the expression of TNFα, IFNγ and IL-4 in CD4+T and CD8+T cells independent of the presented interaction between CD14+monocytes and NKT cells. The described mechanism of n-3 rich lipid emulsions was confirmed in PBMCs from patients with inflammatory bowel disease but not in colorectal cancer patients which seem to lack the interaction between CD14+monocytes and NKT cells. These results show a mechanism for the beneficial effect of the n-3-rich Omegaven in patients with inflammatory conditions but questions its use in patients with cancer. Hence, our results may assist in choosing the best lipid emulsion for patients who require PN.

CD11a regulates hematopoietic stem and progenitor cells.

Integrin αLβ2 (CD11a/CD18, CD11a) is a critical leukocyte adhesion molecule in leukocyte arrest and immunological synapse formation. However, its role in the bone marrow has not been investigated in depth. Here we showed that CD11a was expressed on all subsets of hematopoietic stem and progenitor cells (HPSCs). CD11a deficiency enhanced HSPCs activity under lipopolysaccharide (LPS) stimulation as demonstrated by a higher HSPC cell count along with an increase in cell proliferation. However, our mixed chimera experiment did not support that this phenotype was driven in a cell-intrinsic manner. Rather we found that the production of IL-27, a major cytokine that drives HSPC proliferation, was significantly upregulated both in vivo and in vitro. This adds a novel role of CD11a biology.

Impact of Mycobacterium tuberculosis Glycolipids on the CD4+ T Cell-Macrophage Immunological Synapse.

Mycobacterium tuberculosis cell-wall glycolipids such as mannosylated lipoarabinomannan (ManLAM) can inhibit murine CD4+ T cells by blocking TCR signaling. This results in suppression of IL-2 production, reduced T cell proliferation, and induction of CD4+ T cell anergy. This study extended these findings to the interaction between primary human CD4+ T cells and macrophages infected by mycobacteria. Exposure of human CD4+ T cells to ManLAM before activation resulted in loss of polyfunctionality, as measured by IL-2, IFN-γ, and TNF-α expression, and reduced CD25 expression. This was not associated with upregulation of inhibitory receptors CTLA-4, PD-1, TIM-3, and Lag-3. By confocal microscopy and imaging flow cytometry, ManLAM exposure reduced conjugate formation between macrophages and CD4+ T cells. ManLAM colocalized to the immunological synapse (IS) and reduced translocation of lymphocyte-specific protein tyrosine kinase (LCK) to the IS. When CD4+ T cells and Mycobacterium bovis BCG-infected monocytes were cocultured, ManLAM colocalized to CD4+ T cells, which formed fewer conjugates with infected monocytes. These results demonstrate that mycobacterial cell-wall glycolipids such as ManLAM can traffic from infected macrophages to disrupt productive IS formation and inhibit CD4+ T cell activation, contributing to immune evasion by M. tuberculosis.

Immunological Synapse Formation Between Helper T Cells and Antigen-Presenting Cells

Immunological Synapse Formation Between Helper T Cells and Antigen-Presenting Cells

Immunological Synapse Formation Between Helper T Cells and Antigen-Presenting Cells

Immunological Synapse Formation Between Helper T Cells and Antigen-Presenting Cells

Population dynamics of immunological synapse formation induced by bispecific T cell engagers predict clinical pharmacodynamics and treatment resistance.

Effector T cells need to form immunological synapses (IS) with recognized target cells to elicit cytolytic effects. Facilitating IS formation is the principal pharmacological action of most T cell-based cancer immunotherapies. However, the dynamics of IS formation at the cell population level, the primary driver of the pharmacodynamics of many cancer immunotherapies, remains poorly defined. Using classic immunotherapy CD3/CD19 bispecific T cell engager (BiTE) as our model system, we integrate experimental and theoretical approaches to investigate the population dynamics of IS formation and their relevance to clinical pharmacodynamics and treatment resistance. Our models produce experimentally consistent predictions when defining IS formation as a series of spatiotemporally coordinated events driven by molecular and cellular interactions. The models predict tumor-killing pharmacodynamics in patients and reveal trajectories of tumor evolution across anatomical sites under BiTE immunotherapy. Our models highlight the bone marrow as a potential sanctuary site permitting tumor evolution and antigen escape. The models also suggest that optimal dosing regimens are a function of tumor growth, CD19 expression, and patient T cell abundance, which confer adequate tumor control with reduced disease evolution. This work has implications for developing more effective T cell-based cancer immunotherapies.

Avasimibe can cooperate with a DC-targeting and integration-deficient lentivector to induce stronger HBV specific T cytotoxic response by regulating cholesterol metabolism

We have reported a lentivector which could effectively induce HBV-specific cytotoxic T lymphocytes (CTLs). Avasimibe is an inhibitor of acetyl-CoA acetyltransferase-1 (ACAT1), and has been shown to enhance T lymphocyte cytotoxicity on tumor cells. However, the role of avasimibe in lentivector-induced HBV-specific T cytotoxic response remains unknown. Based on previous study, we constructed an integration-deficient lentivector LVDC-ID-HBV (harboring HBcAg expression), and the in vitro experiments showed that the combination of avasimibe exhibited better efficacy in inducing HBV-specific CTL responses including cell proliferation, production of cytokines, as well as CTL killing activities. Mechanism experiments showed that increasing cell membrane cholesterol levels by MβCD-coated cholesterol or ACAT1 inhibition efficiently promoted TCR clustering, signaling transduction and immunological synapse formation, thereby mediating augmented CTL responses. Nevertheless, the depletion of plasma membrane cholesterol with MβCD led to obviously decreased CTL responses. The avasimibe-mediated strengthened immune effects were also determined in animal experiments and the results were in agreement with those from the in vitro research. In particular, the in vivo CTL killing activities were identified by the CFSE or BV-labeled splenocyte lysis assay. Moreover, the experiments in HBV transgenic mice showed that the LVDC-ID-HBV plus avasimibe group demonstrated the lowest serum HBsAg and HBV DNA levels, as well as the lowest expression of HBsAg and HBcAg in liver tissues. We concluded that the HBV-specific CTL immune responses could be potentiated by avasimibe through regulating plasma membrane cholesterol levels. Avasimibe may be a potential adjuvant for lentivector vaccine against HBV infection.

Role of an adaptor protein human germinal center-associated lymphoma (HGAL) in cell signaling and lymphomagenesis

Human germinal center (GC)-associated lymphoma (HGAL) is a multi-domain adaptor protein expressed in GC B lymphocytes, T follicular helper (Tfh) cells and lymphomas derived from these cells. HGAL expression is an independent predictor of longer survival of diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin’s lymphoma (HL) patients. HGAL regulates B cell receptor (BCR) signaling and immunological synapse formation by binding to either the downstream effectors [e.g., spleen tyrosine kinase (Syk)] or other signaling regulators [e.g., growth factor receptor-bound protein 2 (Grb2)]. HGAL regulates the cytoskeleton that reshapes B cell morphology during BCR signaling and cell motility by at least two molecular mechanisms: enhanced Ras homolog gene family member A (RhoA) signaling and inhibition of myosin-actin translocation. These effects on the cytoskeleton decrease lymphoma dissemination in animal models and contribute to decreased lymphoma dissemination in patients. The latter may contribute to the association of HGAL protein expression with longer survival of patients with DLBCL and HL tumors. The ability to regulate multiple and distinct functions simultaneously in B cells implies that the HGAL protein level is tightly regulated. It was demonstrated that HGAL can be regulated by PR/SET domain 1 (PRDM1)/B lymphocyte-induced maturation protein-1 (BLIMP1) and interleukin-4 (IL-4) at the transcription level, by microRNA-155 (miR-155) at the post-transcriptional level, and by F-box protein 10 (FBXO10) at the post-translational level. Constitutive enforced expression of HGAL at physiological levels leads to lymphoid hyperplasia and DLBCL in mice. Future studies need to focus on identifying HGAL interactome, dissecting its interaction network, and understanding HGAL spatiotemporal signaling in live cells in physiological conditions. Further, the recent demonstration of HGAL expression in Tfh cells requires the determination of its function in these cells. These studies will contribute to new insights into the biology of these cellular subsets and how immune dysregulation contributes to lymphomagenesis.

Endosome Traffic Modulates Pro-Inflammatory Signal Transduction in CD4+ T Cells-Implications for the Pathogenesis of Systemic Lupus Erythematosus.

Endocytic recycling regulates the cell surface receptor composition of the plasma membrane. The surface expression levels of the T cell receptor (TCR), in concert with signal transducing co-receptors, regulate T cell responses, such as proliferation, differentiation, and cytokine production. Altered TCR expression contributes to pro-inflammatory skewing, which is a hallmark of autoimmune diseases, such as systemic lupus erythematosus (SLE), defined by a reduced function of regulatory T cells (Tregs) and the expansion of CD4+ helper T (Th) cells. The ensuing secretion of inflammatory cytokines, such as interferon-γ and interleukin (IL)-4, IL-17, IL-21, and IL-23, trigger autoantibody production and tissue infiltration by cells of the adaptive and innate immune system that induce organ damage. Endocytic recycling influences immunological synapse formation by CD4+ T lymphocytes, signal transduction from crosslinked surface receptors through recruitment of adaptor molecules, intracellular traffic of organelles, and the generation of metabolites to support growth, cytokine production, and epigenetic control of DNA replication and gene expression in the cell nucleus. This review will delineate checkpoints of endosome traffic that can be targeted for therapeutic interventions in autoimmune and other disease conditions.

An engineered Trop2/CD3 bispecific antibody for treatment of gastric, pancreatic, and colorectal cancers.

e14521 Background: Given its over-expression and poor prognosis in a variety of solid tumors, trophoblast cell surface antigen 2 (Trop2) is regarded as a potential therapeutic target. Trop2-directed antibody-drug conjugates have been developed for treatment of solid tumors; however, their clinical benefits can be limited by the associated undesirable effects which is likely attributed to the on-target off-tumor effect arising from the low basal Trop2 expression on the surface of multiple normal epithelial tissues. The present study aims to develop a humanized Trop2/CD3 bispecific T-cell engager antibody that can promote immunological synapse formation with tumor cells overexpressing Trop2, redirecting T cell-mediated killing specifically to cancers with reduced risk of on-target off-tumor effects. Methods: A Trop2/CD3 bispecific antibody was generated by linking a humanized anti-Trop2 monoclonal antibody with a CD3-binding scFv. To tune the affinity of the bispecific antibody for tumor target, in silico paratope prediction was firstly done using ProABC-2 (Ambrosetti F et al., 2020), following by alanine substitution of the identified paratope residues individually. A total of 6 CDR variants were generated with their binding to Trop2 examined using biolayer interferometry. Selected candidates were further characterized for their binding to Trop2-positive cancer cells and activation of T cells upon target engagement. Redirection of T cell-mediated killing on cancer cells expressing high Trop2 level was also studied in vitro. Results: Substitution of a complementarity-determining residue (CDR) on the heavy chain of anti-Trop2 antibody with an alanine significantly reduced binding affinity to Trop2. This CDR variant was selected for further functional characterization. Apart from weaker binding to Trop2-positive tumor cells, the variant also demonstrated a slower cytokine induction, a less acute T-cell activation, and a cytotoxic effect which was more restricted to Trop2-high cancer cells. Conclusions: Our new Trop2/CD3 bispecific antibody demonstrates potent anti-tumor activity, while at the same time shows a reduced risk of on-target off-tumor toxicity. The drug candidate warrants further investigations including toxicity study in monkeys. [Table: see text]

Lipid mediators in the regulation of innate and adaptive immunity.

Lipid mediators in the regulation of innate and adaptive immunity.

Regulation of CD8 T-cell signaling, metabolism, and cytotoxic activity by extracellular lysophosphatidic acid.

Lysophosphatidic acid (LPA) is an endogenous bioactive lipid that is produced extracellularly and signals to cells via cognate LPA receptors, which are G-protein coupled receptors (GPCRs). Mature lymphocytes in mice and humans express three LPA receptors, LPA2 , LPA5, and LPA6 , and work from our group has determined that LPA5 signaling by T lymphocytes inhibits specific antigen-receptor signaling pathways that ultimately impair lymphocyte activation, proliferation, and function. In this review, we discuss previous and ongoing work characterizing the ability of an LPA-LPA5 axis to serve as a peripheral immunological tolerance mechanism that restrains adaptive immunity but is subverted during settings of chronic inflammation. Specifically, LPA-LPA5 signaling is found to regulate effector cytotoxic CD8 T cells by (at least) two mechanisms: (i) regulating the actin-microtubule cytoskeleton in a manner that impairs immunological synapse formation between an effector CD8 T cell and antigen-specific target cell, thus directly impairing cytotoxic activity, and (ii) shifting T-cell metabolism to depend on fatty-acid oxidation for mitochondrial respiration and reducing metabolic efficiency. The in vivo outcome of LPA5 inhibitory activity impairs CD8 T-cell killing and tumor immunity in mouse models providing impetus to consider LPA5 antagonism for the treatment of malignancies and chronic infections.

LFA-1 Activation in T-Cell Migration and Immunological Synapse Formation.

Integrin LFA-1 plays a critical role in T-cell migration and in the formation of immunological synapses. LFA-1 functions through interacting with its ligands with differing affinities: low, intermediate, and high. Most prior research has studied how LFA-1 in the high-affinity state regulates the trafficking and functions of T cells. LFA-1 is also presented in the intermediate-affinity state on T cells, however, the signaling to activate LFA-1 to the intermediate-affinity state and the role of LFA-1 in this affinity state both remain largely elusive. This review briefly summarizes the activation and roles of LFA-1 with varied ligand-binding affinities in the regulation of T-cell migration and immunological synapse formation.