Joseph D. Tario

Tracking antigen‐driven responses by flow cytometry: Monitoring proliferation by dye dilution

Cell‐tracking reagents such as the green‐fluorescent protein labeling dye CFSE and the red‐fluorescent lipophilic membrane dye PKH26 are commonly used to monitor cell proliferation by flow cytometry in heterogeneous cell populations responding to immune stimuli. Both reagents stain cells with a bright homogeneous fluorescence, which is partitioned between daughter cells during each cell division. Because daughter cell fluorescence intensities are approximately halved after each division, the intensity of a cell relative to its intensity at the time of staining provides information about how many divisions it has undergone. Knowing how many rounds of division have occurred and the relative number of cells in each daughter generation, one can back‐calculate the number of cells in the original population (i.e., cells present at the time of stimulus) that went on to respond by proliferating. Using this information, the precursor cell frequencies and extent of expansion to a specific antigen or mitogen of interest can be calculated. Concurrently, the phenotype of the cells can be determined, as well as their ability to bind antigen or synthesize cytokines, providing more detailed characterization of all cells responding to the antigen, not just effector cells. In multiparameter flow cytometric experiments to simultaneously analyze antigen‐specific tetramer binding, cytokine production and T‐cell proliferation, we found that only approximately half of the cells that exhibited specific binding to influenza tetramer also proliferated, as measured by dye dilution, and synthesized IFNγ in response to antigen. We expect the advent of new cell tracking dyes emitting from the violet to the near infrared combined with the increasing number of lasers and detectors on contemporary flow cytometers to further expand the usefulness of this approach to characterization of complex antigen‐driven immunological responses.

Efficacy of Levo-1-Methyl Tryptophan and Dextro-1-Methyl Tryptophan in Reversing Indoleamine-2,3-Dioxygenase-Mediated Arrest of T-Cell Proliferation in Human Epithelial Ovarian Cancer

It has been reported that levo-1-methyl tryptophan (L-1MT) can block indoleamine-2,3-dioxygenase (IDO) expressed by human dendritic cells (DC), whereas dextro-1-methyl tryptophan (D-1MT) is inefficient. However, whether L-1MT or D-1MT can efficiently reverse IDO-induced arrest of human T-cell proliferation has not been clarified. Here, we show a marked immunosuppressive effect of IDO derived from INDO-transfected 293 cell, IDO+ ovarian cancer cells, and monocyte-derived DCs on CD4+ Th1 cells, CD8+ T cells, and natural killer cells derived from peripheral blood, ascites, and tumors of ovarian cancer patients. We found that, whereas L-1MT and D/L-1MT can restore proliferation of tumor-derived and peripheral blood T-cell subsets, D-1MT does not effectively restore IDO-induced arrest of T-cell proliferation. Although D-1MT inhibited kynurenine production at high concentrations, L-1MT was more effective in abrogating kynurenine generation and tryptophan depletion, whereas tryptophan was completely depleted by IDO even in the presence of high amounts of D-1MT. Together, the results indicate that, whereas the generation of tryptophan metabolites (kynurenines) by IDO is important in mediating suppression of T-cell proliferation, the degree to which tryptophan depletion is restored by 1MT is also critical in overcoming IDO-induced arrest of T-cell proliferation.

Toll-like receptor activation enhances cell-mediated immunity induced by an antibody vaccine targeting human dendritic cells

Previously, we have successfully targeted the mannose receptor (MR) expressed on monocyte-derived dendritic cells (DCs) using a fully human MR-specific antibody, B11, as a vehicle to deliver whole protein tumor antigens such as the human chorionic gonadotropin hormone (hCGβ). Since MRs play a role in bridging innate immunity with adaptive immunity we have explored several toll-like receptor (TLR)-specific ligands that may synergize with MR targeting and be applicable as adjuvants in the clinic. We demonstrate that antigen-specific helper and cytolytic T cells from both healthy donors and cancer patients were effectively primed with B11-hCGβ-treated autologous DCs when a combination of one or several TLR ligands is used. Specifically, concomitant signaling of DCs via TLR3 with dsRNA (poly I:C) and DC TLR 7/8 with Resiquimod (R-848), respectively, elicited efficient antigen presentation-mediated by MR-targeting. We demonstrate that MR and TLRs contribute towards maturation and activation of DCs by a mechanism that may be driven by a combination of adjuvant and antibody vaccines that specifically deliver antigenic targets to DCs.

Tracking Immune Cell Proliferation and Cytotoxic Potential Using Flow Cytometry

In the second edition of this series, we described the use of cell tracking dyes in combination with tetramer reagents and traditional phenotyping protocols to monitor levels of proliferation and cytokine production in antigen-specific CD8(+) T cells. In particular, we illustrated how tracking dye fluorescence profiles could be used to ascertain the precursor frequencies of different subsets in the T-cell pool that are able to bind tetramer, synthesize cytokines, undergo antigen-driven proliferation, and/or carry out various combinations of these functional responses.Analysis of antigen-specific proliferative responses represents just one of many functions that can be monitored using cell tracking dyes and flow cytometry. In this third edition, we address issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T-cell functions. We summarize key characteristics of and differences between general protein- and membrane-labeling dyes, discuss determination of optimal staining concentrations, and provide detailed labeling protocols for both dye types. Examples of the advantages of two-color cell tracking are provided in the form of protocols for (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay and (b) simultaneous monitoring of proliferative responses in effector and regulatory T cells.

Flow cytometry and gene expression profiling of immune cells of the carotid plaque and peripheral blood.

OBJECTIVES The relative contribution of the local vs. peripheral inflammation to the atherothrombotic processes is unknown. We compared the inflammatory status of the immune cells of the carotid plaque with similar cells in peripheral circulation of patients with advanced carotid disease (PCDs). METHODS Mononuclear cells (MNCs) were extracted from carotid endarterectomy (CEA) samples by enzymatic digestion and subsequent magnetic cell sorting. The cell surface antigenic expressions, and mRNA expression levels were compared between CEA MNCs and peripheral MNCs, using flow cytometry and RT-PCR techniques. RESULTS The percentages of resting MNCs were lower, and activated MNCs, particularly monocytes, were higher in the CEAMNCs, as compared to the peripheral MNCs. The percentages of activated T cells and B cells were higher in the peripheral MNCs of PCDs, than in healthy controls (HCs), but the percentages of activated monocytes did not differ between the two groups. The expression levels of both pro-inflammatory/pro-thrombotic (P(38), JNKB-1, Egr-1 PAI-1, MCP-1, TF, MMP-9, HMGB-1, TNF-α, mTOR) and anti-inflammatory (PPAR-γ, TGF-β) mediators were significantly higher in the CEA MNCs as compared to the peripheral MNCs. Furthermore, MMP-9 and PPAR-γ expression levels were higher in the peripheral MNCs of PCDs than HCs. CONCLUSION The inflammatory status is higher in the immune cells of the carotid plaque, as compared to those cells in the peripheral blood. The altered expression levels of both pro-inflammatory/pro-thrombotic and anti-inflammatory mediators in the milieu of the plaque suggest that the balance between these various mediators may play a key role in carotid disease progression.

Simultaneous, Single-Cell Measurement of Messenger RNA, Cell Surface Proteins, and Intracellular Proteins.

Nucleic acid content can be quantified by flow cytometry through the use of intercalating compounds; however, measuring the presence of specific sequences has hitherto been difficult to achieve by this methodology. The primary obstacle to detecting discrete nucleic acid sequences by flow cytometry is their low quantity and the presence of high background signals, rendering the detection of hybridized fluorescent probes challenging. Amplification of nucleic acid sequences by molecular techniques such as in situ PCR have been applied to single‐cell suspensions, but these approaches have not been easily adapted to conventional flow cytometry. An alternative strategy implements a Branched DNA technique, comprising target‐specific probes and sequentially hybridized amplification reagents, resulting in a theoretical 8,000‐ to 16,000‐fold increase in fluorescence signal amplification. The Branched DNA technique allows for the quantification of native and unmanipulated mRNA content with increased signal detection and reduced background. This procedure utilizes gentle fixation steps with low hybridization temperatures, leaving the assayed cells intact to permit their concomitant immunophenotyping. This technology has the potential to advance scientific discovery by correlating potentially small quantities of mRNA with many biological measurements at the single‐cell level.

Monitoring Cell Proliferation by Dye Dilution: Considerations for Probe Selection

In the third edition of this series, we described protocols for labeling cell populations with tracking dyes, and addressed issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T cell functions. We summarized key characteristics of and differences between general protein and membrane labeling dyes, discussed determination of optimal staining concentrations, and provided detailed labeling protocols for both dye types. Examples of the advantages of two-color cell tracking were provided in the form of protocols for: (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay; and (b) an in vitro suppression assay for simultaneous proliferation monitoring of effector and regulatory T cells.The number of commercially available fluorescent cell tracking dyes has expanded significantly since the last edition, with new suppliers and/or new spectral properties being added at least annually. In this fourth edition, we describe evaluations to be performed by the supplier and/or user when characterizing a new cell tracking dye and by the user when selecting one for use in multicolor proliferation monitoring. These include methods for: (a) Assessment of the dyes spectral profile on the laboratorys flow cytometer(s) to optimize compatibility with other employed fluorochromes and minimize compensation problems; (b) Evaluating the effect of labeling on cell growth rate;

Diagnosis of Plasma Cell Dyscrasias and Monitoring of Minimal Residual Disease by Multiparametric Flow Cytometry

Plasma cell dyscrasia (PCD) is a heterogeneous disease that has seen a tremendous change in outcomes due to improved therapies. Over the past few decades, multiparametric flow cytometry has played an important role in the detection and monitoring of PCDs. Flow cytometry is a high-sensitivity assay for early detection of minimal residual disease (MRD) that correlates well with progression-free survival and overall survival. Before flow cytometry can be effectively implemented in the clinical setting, sample preparation, panel configuration, analysis, and gating strategies must be optimized to ensure accurate results. Current consensus methods and reporting guidelines for MRD testing are discussed.

Immune signatures associated with improved progression-free and overall survival for myeloma patients treated with AHSCT

Multiple therapeutic options exist for multiple myeloma (MM), including autologous hematopoietic stem cell transplantation (AHSCT). Measurement of minimal residual disease (MRD) and immune reconstitution is rapidly becoming an integral part of the care of MM patients. We investigated comprehensive immune profiling (IP) associated with progression-free survival (PFS) and overall survival (OS). From August 2007 to January 2014, 101 consecutive MM patients underwent peripheral blood IP and marrow MRD testing before and approximately 100 days after AHSCT. Higher pre-AHSCT CD19+ B-cell counts correlated with improved 2-year PFS (83% [highest quartile] vs 53% [lowest quartile]; P = .01) and OS (93% [highest quartile] vs 63% [lowest quartile]; P = .0003). This effect was seen primarily in patients with MRD-positive marrow tests. Higher γδ T-cell counts post-AHSCT correlated with improved 2-year PFS (65% [highest quartile] vs 45% [lowest quartile]; P = .02) and OS (89% [highest quartile] vs 65% [lowest quartile]; P = .01). Higher CD4+ central memory (CM) cell counts post-AHSCT were associated with improved 2-year OS (95% [upper quartile] vs 47% [lowest quartile]; P = .0003) but not PFS. The higher γδ T-cell and CD4+ CM-cell count associations were primarily observed in MRD-negative patients post-AHSCT and in patients not receiving maintenance therapy. This proof-of-concept study demonstrates that IP before and after AHSCT can be of complementary prognostic value for depth of response. Maintenance therapy seems to overcome negative IP. IP and MRD should be measured in clinical trials of maintenance therapy with novel agents post-AHSCT for MM to confirm their utility for prognosis and management.

BMT CTN Myeloma Intergroup Workshop on Minimal Residual Disease and Immune Profiling: Summary and Recommendations From the Organizing Committee

The Blood and Marrow Transplant Clinical Trials Network Myeloma Intergroup Workshop on Minimal Residual Disease and Immune Profiling was convened on December 1, 2016 at the American Society of Hematology meeting to discuss the emerging data and technologies for minimal residual disease assessment and immune profiling in myeloma. Particular emphasis was placed on developing strategies to incorporate these techniques into clinical trial design. This document reviews the literature, summarizes the topics discussed in the workshop, and provides recommendations for integration of these techniques into future clinical trial design.