TrackAnalyzer: A Fiji/ImageJ toolbox for a holistic analysis of tracks.

Current live-cell imaging techniques make possible the observation of live events and the acquisition of large datasets to characterize the different parameters of the visualized events. They provide new insights into the dynamics of biological processes with unprecedented spatial and temporal resolutions. Here we describe the implementation and application of a new tool called TrackAnalyzer, accessible from Fiji and ImageJ. Our tool allows running semi-automated single-particle tracking (SPT) and subsequent motion classification, as well as quantitative analysis of diffusion and intensity for selected tracks relying on the graphical user interface (GUI) for large sets of temporal images (X-Y-T or X-Y-C-T dimensions). TrackAnalyzer also allows 3D visualization of the results as overlays of either spots, cells or end-tracks over time, along with corresponding feature extraction and further classification according to user criteria. Our analysis workflow automates the following steps: (1) spot or cell detection and filtering, (2) construction of tracks, (3) track classification and analysis (diffusion and chemotaxis), and (4) detailed analysis and visualization of all the outputs along the pipeline. All these analyses are automated and can be run in batch mode for a set of similar acquisitions.

CyTOF analysis identifies unusual immune cells in urine of BCG-treated bladder cancer patients.

High grade non-muscle-invasive bladder tumours are treated with transurethral resection followed by recurrent intravesical instillations of Bacillus Calmette Guérin (BCG). Although most bladder cancer patients respond well to BCG, there is no clinical parameter predictive of treatment response, and when treatment fails, the prognosis is very poor. Further, a high percentage of NMIBC patients treated with BCG suffer unwanted effects that force them to stop treatment. Thus, early identification of patients in which BCG treatment will fail is really important. Here, to identify early stage non-invasive biomarkers of non-responder patients and patients at risk of abandoning the treatment, we longitudinally analysed the phenotype of cells released into the urine of bladder cancer patients 3-7 days after BCG instillations. Mass cytometry (CyTOF) analyses revealed a large proportion of granulocytes and monocytes, mostly expressing activation markers. A novel population of CD15+CD66b+CD14+CD16+ cells was highly abundant in several samples; expression of these markers was confirmed using flow cytometry and qPCR. A stronger inflammatory response was associated with increased cell numbers in the urine; this was not due to hematuria because the cell proportions were distinct from those in the blood. This pilot study represents the first CyTOF analysis of cells recruited to urine during BCG treatment, allowing identification of informative markers associated with treatment response for sub-selection of markers to confirm using conventional techniques. Further studies should jointly evaluate cells and soluble factors in urine in larger cohorts of patients to characterise the arms of the immune response activated in responders and to identify patients at risk of complications from BCG treatment.

Sphingomyelin Depletion Inhibits CXCR4 Dynamics and CXCL12-Mediated Directed Cell Migration in Human T Cells.

Sphingolipids, ceramides and cholesterol are integral components of cellular membranes, and they also play important roles in signal transduction by regulating the dynamics of membrane receptors through their effects on membrane fluidity. Here, we combined biochemical and functional assays with single-particle tracking analysis of diffusion in the plasma membrane to demonstrate that the local lipid environment regulates CXCR4 organization and function and modulates chemokine-triggered directed cell migration. Prolonged treatment of T cells with bacterial sphingomyelinase promoted the complete and sustained breakdown of sphingomyelins and the accumulation of the corresponding ceramides, which altered both membrane fluidity and CXCR4 nanoclustering and dynamics. Under these conditions CXCR4 retained some CXCL12-mediated signaling activity but failed to promote efficient directed cell migration. Our data underscore a critical role for the local lipid composition at the cell membrane in regulating the lateral mobility of chemokine receptors, and their ability to dynamically increase receptor density at the leading edge to promote efficient cell migration.

A set point in the selection of the αßTCR T cell repertoire imposed by pre-TCR signaling strength.

Signaling via the T cell receptor (TCR) is critical during the development, maintenance, and activation of T cells. Quantitative aspects of TCR signaling have an important role during positive and negative selection, lineage choice, and ability to respond to small amounts of antigen. By using a mutant mouse line expressing a hypomorphic allele of the CD3ζ chain, we show here that the strength of pre-TCR­mediated signaling during T cell development determines the diversity of the TCRß repertoire available for positive and negative selection, and hence of the final αßTCR repertoire. This finding uncovers an unexpected, pre-TCR signaling­dependent and repertoire­shaping role for ß-selection beyond selection of in-frame rearranged TCRß chains. Our data furthermore support a model of pre-TCR signaling in which the arrangement of this receptor in stable nanoclusters determines its quantitative signaling capacity.

Altered CXCR4 dynamics at the cell membrane impairs directed cell migration in WHIM syndrome patients.

Chemokine receptor nanoscale organization at the cell membrane is orchestrated by the actin cytoskeleton and influences cell responses. Using single-particle tracking analysis we show that CXCR4R334X, a truncated mutant chemokine receptor linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), fails to nanoclusterize after CXCL12 stimulation, and alters the lateral mobility and spatial organization of CXCR4 when coexpressed. These findings correlate with multiple phalloidin-positive protrusions in cells expressing CXCR4R334X, and their inability to correctly sense chemokine gradients. The underlying mechanisms involve inappropriate actin cytoskeleton remodeling due to the inadequate ß-arrestin1 activation by CXCR4R334X, which disrupts the equilibrium between activated and deactivated cofilin. Overall, we provide insights into the molecular mechanisms governing CXCR4 nanoclustering, signaling and cell function, and highlight the essential scaffold role of ß-arrestin1 to support CXCL12-mediated actin reorganization and receptor clustering. These defects associated with CXCR4R334X expression might contribute to the severe immunological symptoms associated with WHIM syndrome.

Single-reaction multi-antigen serological test for comprehensive evaluation of SARS-CoV-2 patients by flow cytometry.

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19.

The multilayered complexity of the chemokine receptor system.

The chemokines receptor family are membrane-expressed class A-specific seven-transmembrane receptors linked to G proteins. Through interaction with the corresponding ligands, the chemokines, they induce a wide variety of cellular responses including cell polarization, movement, immune and inflammatory responses, as well as the prevention of HIV-1 infection. Like a Russian matryoshka doll, the chemokine receptor system is more complex than initially envisaged. This review focuses on the mechanisms that contribute to this dazzling complexity and how they modulate the signaling events triggered by chemokines. The chemokines and their receptors exist as monomers, dimers and oligomers, their expression pattern is highly regulated, and the ligands can bind distinct receptors with similar affinities. The use of novel imaging-based technologies, particularly real-time imaging modalities, has shed new light on the very dynamic conformations that chemokine receptors adopt depending on the cellular context, and that affect chemokine-mediated responses. This complex scenario presents both challenging and exciting opportunities for drug discovery.

The Role of the CXCL12/CXCR4/ACKR3 Axis in Autoimmune Diseases.

Chemokine receptors are members of the G protein-coupled receptor superfamily. These receptors are intimately involved in cell movement, and thus play a critical role in several physiological and pathological situations that require the precise regulation of cell positioning. CXCR4 is one of the most studied chemokine receptors and is involved in many functions beyond leukocyte recruitment. During embryogenesis, it plays essential roles in vascular development, hematopoiesis, cardiogenesis, and nervous system organization. It has been also implicated in tumor progression and autoimmune diseases and, together with CD4, is one of the co-receptors used by the HIV-1 virus to infect immune cells. In contrast to other chemokine receptors that are characterized by ligand promiscuity, CXCR4 has a unique ligand-stromal cell-derived factor-1 (SDF1, CXCL12). However, this ligand also binds ACKR3, an atypical chemokine receptor that modulates CXCR4 functions and is overexpressed in multiple cancer types. The CXCL12/CXCR4/ACKR3 axis constitutes a potential therapeutic target for a wide variety of inflammatory diseases, not only by interfering with cell migration but also by modulating immune responses. Thus far, only one antagonist directed against the ligand-binding site of CXCR4, AMD3100, has demonstrated clinical relevance. Here, we review the role of this ligand and its receptors in different autoimmune diseases.

BCG Therapy of Bladder Cancer Stimulates a Prolonged Release of the Chemoattractant CXCL10 (IP10) in Patient Urine.

Background: Intra-vesical instillation of Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is an effective therapy for high-grade non-muscle invasive bladder cancer (NMIBC), which provokes a local immune response resulting in 70% of patients free of relapse after three years. Because non-responder patients usually have a bad prognosis, the early identification of treatment failure is crucial. We hypothesized that, if an effective immune response was taking place in the bladder, soluble factors would be released to the urine many days after BCG instillations. Methods: An extensive panel of cytokines and chemokines released into the urine seven days after every BCG instillation was screened in a cohort of NMIBC patients over three years. Results: The determinations of the urinary concentrations of cytokines, chemokines, and creatinine showed that increasing concentrations of C-X-C motif chemokine 10 (CXCL10) also known as interferon-inducible protein 10 (IP10) could be detected during the six-week induction cycle of BCG-treated patients released into the urine by CD14+ cells. In vitro, CXCL10 facilitated the recruitment of effector immune cells after the BCG-mediated upregulation of CXCR3 in both T- and natural killer (NK)-cells. Conclusions: The high concentrations of chemokine detected one week after the encounter with mycobacteria suggest that the CXCL10 axis might be related to the intensity of the immune anti-tumor response.

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy.

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Inhibitory Role of Growth Hormone in the Induction and Progression Phases of Collagen-Induced Arthritis.

Evidence indicates an intimate connection between the neuroendocrine and the immune systems. A number of in vitro and in vivo studies have demonstrated growth hormone (GH) involvement in immune regulation. The GH receptor is expressed by several leukocyte subpopulations, and GH modulates immune cell proliferation and activity. Here, we found that sustained GH expression protected against collagen-induced arthritis (CIA); in GH-transgenic C57BL/6 (GHTg) mice, disease onset was delayed, and its overall severity was decreased. The anti-collagen response was impaired in these mice, as were inflammatory cytokine levels. Compared to control arthritic littermates, immunized GHTg mice showed significantly lower RORγt (retinoic acid receptor-related orphan receptor gamma 2), IL-17, GM-CSF, IL-22, and IFNγ mRNA expression in draining lymph nodes, whereas there were no differences in IL-21, IL-6, or IL-2 mRNA levels. Data thus suggest that Th17/Th1 cell plasticity toward a pathological phenotype is reduced in these mice. Exogenous GH administration in arthritic DBA/1J mice reduced the severity of established CIA as well as the inflammatory environment, which also shows a GH effect on arthritis progression. These results indicate that GH prevents inflammatory joint destruction in CIA. Our findings demonstrate a modulatory GH role in immune system function that contributes to alleviating CIA symptoms and underlines the importance of endocrine regulation of the immune response.

Separating Actin-Dependent Chemokine Receptor Nanoclustering from Dimerization Indicates a Role for Clustering in CXCR4 Signaling and Function.

A current challenge in cell motility studies is to understand the molecular and physical mechanisms that govern chemokine receptor nanoscale organization at the cell membrane, and their influence on cell response. Using single-particle tracking and super-resolution microscopy, we found that the chemokine receptor CXCR4 forms basal nanoclusters in resting T cells, whose extent, dynamics, and signaling strength are modulated by the orchestrated action of the actin cytoskeleton, the co-receptor CD4, and its ligand CXCL12. We identified three CXCR4 structural residues that are crucial for nanoclustering and generated an oligomerization-defective mutant that dimerized but did not form nanoclusters in response to CXCL12, which severely impaired signaling. Overall, our data provide new insights to the field of chemokine biology by showing that receptor dimerization in the absence of nanoclustering is unable to fully support CXCL12-mediated responses, including signaling and cell function in vivo.

Impaired NK cell recognition of vemurafenib-treated melanoma cells is overcome by simultaneous application of histone deacetylase inhibitors.

Therapy of metastatic melanoma advanced recently with the clinical implementation of signalling pathway inhibitors, such as vemurafenib, specifically targeting mutant BRAFV600E. In general, patients experience remarkable clinical responses under BRAF inhibitor (BRAFi) treatment but eventually progress within 6-8 months due to resistance development. Responding metastases show an increased immune cell infiltrate, including also NK cells, that, however, is no longer detectable in BRAFi-resistant lesions, suggesting NK cell activity should be exploited to prevent disease progression. Here, we examined the effects of BRAFi on the expression of ligands targeting activating NK cells receptors immediately after treatment onset, prior to resistance development. We demonstrate that BRAFV600E mutant melanoma cells cultured in the presence of vemurafenib, strongly decreased surface expression of ligands for NK activating receptors including the NKG2D-ligand, MICA, and the DNAM-1 ligand, CD155, and became significantly less susceptible to NK cell attack. NKG2D-ligand protein downregulation was due to a significant decrease in mRNA levels, already detectable 24 h after drug treatment. Interestingly, vemurafenib-induced MICA downregulation could be counteracted by treatment of melanoma cells with the histone deacetylase (HDAC) inhibitor (HDACi) sodium butyrate, that also upregulated the DNAM1-ligand, Nectin-2. HDACi treatment enhanced surface expression of NKG2D-ligands in the presence of BRAFi, accompanied by recovery of NK cell recognition, but only upon simultaneous drug application. These results suggest that co-administration of BRAFi and HDAC inhibitors as well as having direct effects on melanoma cell survival, could also synergise to improve NK cell recognition and avoid tumour immune evasion.

Characterization of a human anti-tumoral NK cell population expanded after BCG treatment of leukocytes.

Immunotherapy, via intra-vesical instillations of BCG, is the therapy of choice for patients with high-risk non-muscle invasive bladder cancer. The subsequent recruitment of lymphocytes and myeloid cells, as well as the release of cytokines and chemokines, is believed to induce a local immune response that eliminates these tumors, but the detailed mechanisms of action of this therapy are not well understood. Here, we have studied the phenotype and function of the responding lymphocyte populations as well as the spectrum of cytokines and chemokines produced in an in vitro model of human peripheral blood mononuclear cells (PBMCs) co-cultured with BCG. Natural killer (NK) cell activation was a prominent feature of this immune response and we have studied the expansion of this lymphocyte population in detail. We show that, after BCG stimulation, CD56dim NK cells proliferate, upregulate CD56, but maintain the expression of CD16 and the ability to mediate ADCC. CD56bright NK cells also contribute to this expansion by increasing CD16 and KIR expression. These unconventional CD56bright cells efficiently degranulated against bladder cancer cells and the expansion of this population required the release of soluble factors by other immune cells in the context of BCG. Consistent with these in vitro data, a small, but significant increase in the intensity of CD16 expression was noted in peripheral blood CD56bright cells from bladder cancer patients undergoing BCG therapy, that was not observed in patients treated with mitomycin-C instillations. These observations suggest that activation of NK cells may be an important component of the anti-tumoral immune response triggered by BCG therapy in bladder cancer.

Ionomycin Treatment Renders NK Cells Hyporesponsive.

Natural killer cells are cytotoxic lymphocytes important in immune responses to cancer and multiple pathogens. However, chronic activation of NK cells can induce a hyporesponsive state. The molecular basis of the mechanisms underlying the generation and maintenance of this hyporesponsive condition are unknown, thus an easy and reproducible mechanism able to induce hyporesponsiveness on human NK cells would be very useful to gain understanding of this process. Human NK cells treated with ionomycin lose their ability to degranulate and secrete IFN-γ in response to a variety of stimuli, but IL-2 stimulation can compensate these defects. Apart from reductions in the expression of CD11a/CD18, no great changes were observed in the activating and inhibitory receptors expressed by these NK cells, however their transcriptional signature is different to that described for other hyporesponsive lymphocytes.

Transfer of the human NKG2D ligands UL16 binding proteins (ULBP) 1-3 is related to lytic granule release and leads to ligand retransfer and killing of ULBP-recipient natural killer cells.

After immune interactions, membrane fragments can be transferred between cells. This fast transfer of molecules is transient and shows selectivity for certain proteins; however, the constraints underlying acquisition of a protein are unknown. To characterize the mechanism and functional consequences of this process in natural killer (NK) cells, we have compared the transfer of different NKG2D ligands. We show that human NKG2D ligands can be acquired by NK cells with different efficiencies. The main findings are that NKG2D ligand transfer is related to immune activation and receptor-ligand interaction and that NK cells acquire these proteins during interactions with target cells that lead to degranulation. Our results further demonstrate that NK cells that have acquired NKG2D ligands can stimulate activation of autologous NK cells. Surprisingly, NK cells can also re-transfer the acquired molecule to autologous effector cells during this immune recognition that leads to their death. These data demonstrate that transfer of molecules occurs as a consequence of immune recognition and imply that this process might play a role in homeostatic tuning-down of the immune response or be used as marker of interaction.