Hijacked Immune Cells in the Tumor Microenvironment: Molecular Mechanisms of Immunosuppression and Cues to Improve T Cell-Based Immunotherapy of Solid Tumors.

The understanding of the tumor microenvironment (TME) has been expanding in recent years in the context of interactions among different cell types, through direct cell-cell communication as well as through soluble factors. It has become evident that the development of a successful antitumor response depends on several TME factors. In this context, the number, type, and subsets of immune cells, as well as the functionality, memory, and exhaustion state of leukocytes are key factors of the TME. Both the presence and functionality of immune cells, in particular T cells, are regulated by cellular and soluble factors of the TME. In this regard, one fundamental reason for failure of antitumor responses is hijacked immune cells, which contribute to the immunosuppressive TME in multiple ways. Specifically, reactive oxygen species (ROS), metabolites, and anti-inflammatory cytokines have central roles in generating an immunosuppressive TME. In this review, we focused on recent developments in the immune cell constituents of the TME, and the micromilieu control of antitumor responses. Furthermore, we highlighted the current challenges of T cell-based immunotherapies and potential future strategies to consider for strengthening their effectiveness.

LFA-1 cluster formation in T-cells depends on L-plastin phosphorylation regulated by P90RSK and PP2A.

The integrin LFA-1 is crucial for T-cell/ APC interactions and sensitive recognition of antigens. Precise nanoscale organization and valency regulation of LFA-1 are mandatory for an appropriate function of the immune system. While the inside-out signals regulating the LFA-1 affinity are well described, the molecular mechanisms controlling LFA-1 avidity are still not fully understood. Here, we show that activation of the actin-bundling protein L-plastin (LPL) through phosphorylation at serine-5 enables the formation of clusters containing LFA-1 in high-affinity conformation. Phosphorylation of LPL is induced by an nPKC-MEK-p90RSK pathway and counter-regulated by the serine-threonine phosphatase PP2A. Interestingly, recruitment of LFA-1 into the T-cell/APC contact zone is not affected by LPL phosphorylation. Instead, for this process, activation of the actin-remodeling protein cofilin through dephosphorylation is essential. Together, this study reveals a dichotomic spatial regulation of LFA-1 clustering and microscale movement in T-cells by two different actin-binding proteins, LPL and cofilin.

Reactive Oxidative Species-Modulated Ca2+ Release Regulates ß2 Integrin Activation on CD4+ CD28null T Cells of Acute Coronary Syndrome Patients.

The number and activity of T cell subsets in the atherosclerotic plaques are critical for the prognosis of patients with acute coronary syndrome. ß2 Integrin activation is pivotal for T cell recruitment and correlates with future cardiac events. Despite this knowledge, differential regulation of adhesiveness in T cell subsets has not been explored yet. In this study, we show that in human T cells, SDF-1α-mediated ß2 integrin activation is driven by a, so far, not-described reactive oxidative species (ROS)-regulated calcium influx. Furthermore, we show that CD4+CD28null T cells represent a highly reactive subset showing 25-fold stronger ß2 integrin activation upon SDF-1α stimulation compared with CD28+ T cells. Interestingly, ROS-dependent Ca release was much more prevalent in the pathogenetically pivotal CD28null subset compared with the CD28+ T cells, whereas the established mediators of the classical pathways for ß2 integrin activation (PKC, PI3K, and PLC) were similarly activated in both T cell subsets. Thus, interference with the calcium flux attenuates spontaneous adhesion of CD28null T cells from acute coronary syndrome patients, and calcium ionophores abolished the observed differences in the adhesion properties between CD28+ and CD28null T cells. Likewise, the adhesion of these T cell subsets was indistinguishable in the presence of exogenous ROS/H2O2 Together, these data provide a molecular explanation of the role of ROS in pathogenesis of plaque destabilization.

Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells.

Several antitumor therapies work by increasing reactive oxygen species (ROS) within the tumor micromilieu. Here, we reveal that L-plastin (LPL), an established tumor marker, is reversibly regulated by ROS-induced thiol oxidation on Cys101, which forms a disulfide bridge with Cys42. LPL reduction is mediated by the Thioredoxin1 (TRX1) system, as shown by TRX1 trapping, TRX1 knockdown and blockade of Thioredoxin1 reductase (TRXR1) with auranofin. LPL oxidation diminishes its actin-bundling capacity. Ratiometric imaging using an LPL-roGFP-Orp1 fusion protein and a dimedone-based proximity ligation assay (PLA) reveal that LPL oxidation occurs primarily in actin-based cellular extrusions and strongly inhibits cell spreading and filopodial extension formation in tumor cells. This effect is accompanied by decreased tumor cell migration, invasion and extracellular matrix (ECM) degradation. Since LPL oxidation occurs following treatment of tumors with auranofin or γ-irradiation, it may be a molecular mechanism contributing to the effectiveness of tumor treatment with redox-altering therapies.

Sulforaphane Inhibits Inflammatory Responses of Primary Human T-Cells by Increasing ROS and Depleting Glutathione.

The activity and function of T-cells are influenced by the intra- and extracellular redox milieu. Oxidative stress induces hypo responsiveness of untransformed T-cells. Vice versa increased glutathione (GSH) levels or decreased levels of reactive oxygen species (ROS) prime T-cell metabolism for inflammation, e.g., in rheumatoid arthritis. Therefore, balancing the T-cell redox milieu may represent a promising new option for therapeutic immune modulation. Here we show that sulforaphane (SFN), a compound derived from plants of the Brassicaceae family, e.g., broccoli, induces a pro-oxidative state in untransformed human T-cells of healthy donors or RA patients. This manifested as an increase of intracellular ROS and a marked decrease of GSH. Consistently, increased global cysteine sulfenylation was detected. Importantly, a major target for SFN-mediated protein oxidation was STAT3, a transcription factor involved in the regulation of TH17-related genes. Accordingly, SFN significantly inhibited the activation of untransformed human T-cells derived from healthy donors or RA patients, and downregulated the expression of the transcription factor RORγt, and the TH17-related cytokines IL-17A, IL-17F, and IL-22, which play a major role within the pathophysiology of many chronic inflammatory/autoimmune diseases. The inhibitory effects of SFN could be abolished by exogenously supplied GSH and by the GSH replenishing antioxidant N-acetylcysteine (NAC). Together, our study provides mechanistic insights into the mode of action of the natural substance SFN. It specifically exerts TH17 prone immunosuppressive effects on untransformed human T-cells by decreasing GSH and accumulation of ROS. Thus, SFN may offer novel clinical options for the treatment of TH17 related chronic inflammatory/autoimmune diseases such as rheumatoid arthritis.

Protein Phosphatase 1α and Cofilin Regulate Nuclear Translocation of NF-κB and Promote Expression of the Anti-Inflammatory Cytokine Interleukin-10 by T Cells.

While several protein serine/threonine kinases control cytokine production by T cells, the roles of serine/threonine phosphatases are largely unexplored. Here, we analyzed the involvement of protein phosphatase 1α (PP1α) in cytokine synthesis following costimulation of primary human T cells. Small interfering RNA (siRNA)-mediated knockdown of PP1α (PP1KD) or expression of a dominant negative PP1α (D95N-PP1) drastically diminished interleukin-10 (IL-10) production. Focusing on a key transcriptional activator of human IL-10, we demonstrate that nuclear translocation of NF-κB was significantly inhibited in PP1KD or D95N-PP1 cells. Interestingly, knockdown of cofilin, a known substrate of PP1 containing a nuclear localization signal, also prevented nuclear accumulation of NF-κB. Expression of a constitutively active nonphosphorylatable S3A-cofilin in D95N-PP1 cells restored nuclear translocation of NF-κB and IL-10 expression. Subpopulation analysis revealed that defective nuclear translocation of NF-κB was most prominent in CD4+ CD45RA- CXCR3- T cells that included IL-10-producing TH2 cells. Together these findings reveal novel functions for PP1α and its substrate cofilin in T cells namely the regulation of the nuclear translocation of NF-κB and promotion of IL-10 production. These data suggest that stimulation of PP1α could limit the overwhelming immune responses seen in chronic inflammatory diseases.

NF-κB inducing kinase (NIK) is an essential post-transcriptional regulator of T-cell activation affecting F-actin dynamics and TCR signaling.

NF-κB inducing kinase (NIK) is the key protein of the non-canonical NF-κB pathway and is important for the development of lymph nodes and other secondary immune organs. We elucidated the specific role of NIK in T cells using T-cell specific NIK-deficient (NIKΔT) mice. Despite showing normal development of lymphoid organs, NIKΔT mice were resistant to induction of CNS autoimmunity. T cells from NIKΔT mice were deficient in late priming, failed to up-regulate T-bet and to transmigrate into the CNS. Proteomic analysis of activated NIK-/- T cells showed de-regulated expression of proteins involved in the formation of the immunological synapse: in particular, proteins involved in cytoskeleton dynamics. In line with this we found that NIK-deficient T cells were hampered in phosphorylation of Zap70, LAT, AKT, ERK1/2 and PLCγ upon TCR engagement. Hence, our data disclose a hitherto unknown function of NIK in T-cell priming and differentiation.

Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8+ T-cells.

The clearance of tumors or virus infected cells is a crucial task of the immune system. Cytotoxic T-cells (CTLs) are able to detect and to kill such altered host cells. Given the recent success of checkpoint inhibitors for tumor therapy, it becomes more and more important to understand the biology of T-cell mediated target cell killing. Tests that allow analyzing the biology of CTLs are either based on flow cytometry or fluorescence microscopy. Thus, they either lack image-based information or have a poor statistical robustness. Therefore, we describe an approach to quantify CTL-mediated cytotoxicity using imaging flow cytometry. Using activated primary human cytotoxic T-cells as CTLs and P815 as target cells, we show that both the evaluation of target cell death and the biology of CTLs can be evaluated in parallel. This enables to gain information about CTL-mediated cytotoxicity in samples from patients important for translational medicine. J. Cell. Biochem. 118: 2528-2533, 2017. © 2017 Wiley Periodicals, Inc.

Qualitative and quantitative analysis of PMN/T-cell interactions by InFlow and super-resolution microscopy.

Neutrophils or polymorphonuclear cells (PMN) eliminate bacteria via phagocytosis and/or NETosis. Apart from these conventional roles, PMN also have immune-regulatory functions. They can transdifferentiate and upregulate MHCII as well as ligands for costimulatory receptors which enables them to behave as antigen presenting cells (APC). The initial step for activating T-cells is the formation of an immune synapse between T-cells and antigen-presenting cells. However, the immune synapse that develops at the PMN/T-cell contact zone is as yet hardly investigated due to the non-availability of methods for analysis of large number of PMN interactions. In order to overcome these obstacles, we introduce here a workflow to analyse the immune synapse of primary human PMN and T-cells using multispectral imaging flow cytometry (InFlow microscopy) and super-resolution microscopy. For that purpose, we used CD3 and CD66b as the lineage markers for T-cells and PMN, respectively. Thereafter, we applied and critically discussed various "masks" for identification of T-cell PMN interactions. Using this approach, we found that a small fraction of transdifferentiated PMN (CD66b+CD86high) formed stable PMN/T-cell conjugates. Interestingly, while both CD3 and CD66b accumulation in the immune synapse was dependent on the maturation state of the PMN, only CD3 accumulation was greatly enhanced by the presence of superantigen. The actin cytoskeleton was weakly rearranged at the PMN side on the immune synapse upon contact with a T-cell in the presence of superantigen. A more detailed analysis using super-resolution microscopy (structured-illumination microscopy, SIM) confirmed this finding. Together, we present an InFlow microscopy based approach for the large scale analysis of PMN/T-cell interactions and - combined with SIM - a possibility for an in-depth analysis of protein translocation at the site of interactions.

Multiparametric Characterization of Human T-Cell Immune Synapses by InFlow Microscopy.

Immune cells need to communicate with each other via direct cell contact formation. The contact zone has similar functions as a neuronal synapse and is therefore named immune synapse. Supramolecular activation clusters consisting of a variety of surface receptors and cytoplasmic proteins are formed within the immune synapse, which are pivotal for T-cell activation. Thus, a malfunction of immune synapse formation has detrimental effects on the healthiness of the individual.Classical confocal microscopy to analyze the supramolecular cluster formation and maturation of the immune synapse between primary human T-cells and antigen-presenting cells is time consuming and the number of cells that can be analyzed is limited. Therefore, we have established an InFlow microscopy approach for the analysis of immune synapses. InFlow microscopy is a hybrid method combining fluorescence microscopy and flow cytometry. Our InFlow microscopy method allows quantifying protein distribution in immune synapses of several hundred or even thousand cell couples in one sample. Importantly, comparisons of different samples with a strong statistical power are possible with InFlow microcopy.

Expression of the bitter receptor T2R38 in pancreatic cancer: localization in lipid droplets and activation by a bacteria-derived quorum-sensing molecule.

T2R38 belongs to the family of bitter receptors and was initially detected in cells of the oral cavity. We now describe expression of T2R38 in tumor cells in patients with pancreatic cancer and in tumor-derived cell lines. T2R38 is localized predominantly intracellular in association with lipid droplets, particularly with the lipid droplet membrane. The receptor can be activated by the bona fide ligand for T2R38, phenylthiourea (PTU), and by N-acetyl-dodecanoyl homoserine (AHL-12), a quorum sensing molecule of Pseudomonas aeruginosa, the latter is the only known natural ligand for T2R38. In response to PTU or AHL-12, key transcription factors are activated including phosphorylation of the MAP kinases p38 and ERK1/2, and upregulation of NFATc1. Moreover, we found increased expression of the multi-drug resistance protein 1 (also known as ABCB1), a transmembrane transporter molecule, participating in shuttling of a plethora of drugs, such as chemotherapeutics or antibiotics. In conclusion, our data indicate a new, additional function of the taste receptor T2R38 beyond sensing "bitter". Moreover, because T2R38 can be stimulated by a bacteria-derived signaling molecule the receptor could link microbiota and cancer.

Tasting Pseudomonas aeruginosa Biofilms: Human Neutrophils Express the Bitter Receptor T2R38 as Sensor for the Quorum Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone.

Bacteria communicate with one another via specialized signaling molecules, known as quorum sensing molecules or autoinducers. The Pseudomonas aeruginosa-derived quorum sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (AHL-12), however, also activates mammalian cells. As shown previously, AHL-12-induced chemotaxis, up-regulated CD11b expression, and enhanced phagocytosis of polymorphonuclear neutrophils. Circumstantial evidence concurred with a receptor for AHL-12, which has been elusive so far. We now investigated the bitter receptor T2R38 as a potential candidate. Although identified as a taste receptor, extragustatory cells express T2R38, for example, epithelial cells in the lung. We now detected T2R38 in peripheral blood neutrophils, monocytes, and lymphocytes. T2R38 is not only found on the cell membrane but also intracellular. In neutrophils, T2R38 was located in vesicles with characteristics of lipid droplets, and super-resolution microscopy showed a co-localization with the lipid droplet membrane. Neutrophils take up AHL-12, and it co-localized with T2R38 as seen by laser scan microscopy. Binding of AHL-12 to T2R28 was confirmed by pull-down assays using biotin-coupled AHL-12 as bait. A commercially available antibody to T2R38 inhibited binding of AHL-12 to neutrophils, and this antibody by itself stimulated neutrophils, similarly to AHL-12. In conclusion, our data provide evidence for expression of functional T2R38 on neutrophils, and are compatible with the notion that T2R38 is the receptor for AHL-12.

InFlow microscopy of human leukocytes: A tool for quantitative analysis of actin rearrangements in the immune synapse.

The actin cytoskeleton is a main component to preserve the cell shape. It represents a cellular machinery that enables morphological changes and orchestrates important dynamic cellular functions. Thereby, it supports T-cell migration, immune synapse formation, activation and execution of effector functions. The analysis of actin rearrangements in T-cells is therefore an important field of basic and clinical research. Actin reorganization is traditionally performed using flow cytometry or confocal microscopy. However, while flow cytometry lacks spatial and structural information, confocal microscopy is time consuming and not feasible for the characterization of rare events or of un-purified primary cell populations. Here we describe a methodology to analyze actin rearrangements using InFlow microscopy, which is a hybrid technique consisting of flow cytometric and microscopic features. We show that InFlow microscopy is a valuable tool for quantification of the amount and distribution of F-actin in human T-cells after stimulation with chemokines or antigen-presenting cells.

Metastasis of prostate cancer and melanoma cells in a preclinical in vivo mouse model is enhanced by L-plastin expression and phosphorylation.

BACKGROUND: Tumor cell migration and metastasis require dynamic rearrangements of the actin cytoskeleton. Interestingly, the F-actin cross-linking and stabilizing protein L-plastin, originally described as a leukocyte specific protein, is aberrantly expressed in several non-hematopoietic malignant tumors. Therefore, it has been discussed as a tumor marker. However, systematic in vivo analyses of the functional relevance of L-plastin for tumor cell metastasis were so far lacking. METHODS: We investigated the relevance of L-plastin expression and phosphorylation by ectopical expression of L-plastin in human melanoma cells (MV3) and knock-down of endogenous L-plastin in prostate cancer (PC3M). The growth and metastatic potential of tumor cells expressing no L-plastin, phosphorylatable or non-phosphorylatable L-plastin was analyzed in a preclinical mouse model after subcutaneous and intracardial injection of the tumor cells. RESULTS: Knock-down of endogenous L-plastin in human prostate carcinoma cells led to reduced tumor cell growth and metastasis. Vice versa, and in line with these findings, ectopic expression of L-plastin in L-plastin negative melanoma cells significantly increased the number of metastases. Strikingly, the metastasis promoting effect of L-plastin was not observed if a non-phosphorylatable L-plastin mutant was expressed. CONCLUSIONS: Our data provide the first in vivo evidence that expression of L-plastin promotes tumor metastasis and, importantly, that this effect depends on an additionally required phosphorylation of L-plastin. In conclusion, these findings imply that for determining the importance of tumor-associated proteins like L-plastin a characterization of posttranslational modifications is indispensable.

Cofilin: a redox sensitive mediator of actin dynamics during T-cell activation and migration.

Cofilin is an actin-binding protein that depolymerizes and/or severs actin filaments. This dual function of cofilin makes it one of the major regulators of actin dynamics important for T-cell activation and migration. The activity of cofilin is spatio-temporally regulated. Its main control mechanisms comprise a molecular toolbox of phospho-, phospholipid, and redox regulation. Phosphorylated cofilin is inactive and represents the dominant cofilin fraction in the cytoplasm of resting human T cells. A fraction of dephosphorylated cofilin is kept inactive at the plasma membrane by binding to phosphatidylinositol 4,5-bisphosphate. Costimulation via the T-cell receptor/CD3 complex (signal 1) together with accessory receptors (signal 2) or triggering through the chemokine SDF1α (stromal cell-derived factor 1α) induce Ras-dependent dephosphorylation of cofilin, which is important for immune synapse formation, T-cell activation, and T-cell migration. Recently, it became evident that cofilin is also highly sensitive for microenvironmental changes, particularly for alterations in the redox milieu. Cofilin is inactivated by oxidation, provoking T-cell hyporesponsiveness or necrotic-like programmed cell death. In contrast, in a reducing environment, even phosphatidylinositol 4,5-bisphosphate-bound cofilin becomes active, leading to actin dynamics in the vicinity of the plasma membrane. In addition to the well-established three signals for T-cell activation, this microenvironmental control of cofilin delivers a modulating signal for T-cell-dependent immune reactions. This fourth modulating signal highly impacts both initial T-cell activation and the effector phase of T-cell-mediated immune responses.

A reducing milieu renders cofilin insensitive to phosphatidylinositol 4,5-bisphosphate (PIP2) inhibition.

Oxidative stress can lead to T cell hyporesponsiveness. A reducing micromilieu (e.g. provided by dendritic cells) can rescue T cells from such oxidant-induced dysfunction. However, the reducing effects on proteins leading to restored T cell activation remained unknown. One key molecule of T cell activation is the actin-remodeling protein cofilin, which is dephosphorylated on serine 3 upon T cell costimulation and has an essential role in formation of mature immune synapses between T cells and antigen-presenting cells. Cofilin is spatiotemporally regulated; at the plasma membrane, it can be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show by NMR spectroscopy that a reducing milieu led to structural changes in the cofilin molecule predominantly located on the protein surface. They overlapped with the PIP2- but not actin-binding sites. Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state. However, it did prevent inhibition of cofilin activity through PIP2. Therefore, a reducing milieu may generate an additional pool of active cofilin at the plasma membrane. Consistently, in-flow microscopy revealed increased actin dynamics in the immune synapse of untransformed human T cells under reducing conditions. Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP2 inhibition, resulting in enhanced actin dynamics.

Depletion of dendritic cells enhances innate anti-bacterial host defense through modulation of phagocyte homeostasis.

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.

L-plastin phosphorylation: a novel target for the immunosuppressive drug dexamethasone in primary human T cells.

Activation of naïve T cells requires costimulation via TCR/CD3 plus accessory receptors, which enables the dynamic rearrangement of the actin cytoskeleton and immune synapse maturation. Signaling events induced following costimulation may thus be valuable targets for therapeutic immunosuppression. Phosphorylation of the actin-bundling protein L-plastin represents such a costimulatory signal in primary human T cells. Phosphorylated L-plastin has a higher affinity toward F-actin. However, the importance of the L-plastin phosphorylation for actin cytoskeleton regulation upon antigen recognition remained unclear. Here, we demonstrate that phosphorylation of L-plastin is important for immune synapse maturation. Thus, expression of nonphosphorylatable L-plastin in untransformed human peripheral blood T cells leads to reduced accumulation of LFA-1 in the immune synapse and to a diminished F-actin increase upon T-cell activation. Interestingly, L-plastin phosphorylation is inhibited by the glucocorticoid dexamethasone. In line with this finding, dexamethasone treatment leads to a reduced F-actin content in stimulated T cells and prevents maturation of the immune synapse. This inhibitory effect of dexamethasone could be reverted by expression of a phospho-mimicking L-plastin mutant. In conclusion, our data introduce costimulation-induced L-plastin phosphorylation as an important event for immune synapse formation and its inhibition by dexamethasone as a novel mode of function of this immunosuppressive glucocorticoid.

Single cell force spectroscopy of T cells recognizing a myelin-derived peptide on antigen presenting cells.

T-cell recognition of peptide-MHC complexes on APCs requires cell-cell interactions. The molecular events leading to T-cell activation have been extensively investigated, but the underlying physical binding forces between T-cells and APCs are largely unknown. We used single cell force spectroscopy for quantitation of interaction forces between T-cells and APCs presenting a tolerogenic peptide derived from myelin basic protein. When T-cells were brought into contact with peptide-loaded APCs, interaction forces increased with time from about 0.5nN after 10s interaction to about 15nN after 30min. In the absence of antigen, or when ICAM-1-negative APC was used, no increase in binding forces was observed. The temporal development of interaction forces correlated with the kinetics of immune synapse formation, as determined by LFA-1 and TCR enrichment at the interface of T-cell/APC conjugates using high throughput multispectral imaging flow cytometry. Together, these results suggest that ICAM-1/LFA-1 redistribution to the contact area is mainly responsible for development of strong interaction forces. High forces will keep T-cells and APCs in tight contact, thereby providing a platform for optimal interaction between TCRs and peptide-MHC complexes.