[Show abstract][Hide abstract] ABSTRACT: Flow cytometric analysis enables the simultaneous single cell interrogation of multiple biomarkers for phenotypic and functional identification of heterogeneous populations. Analysis of polychromatic data has become increasingly complex with more measured parameters. Furthermore, manual gating of multiple populations using standard analysis techniques can lead to errors in data interpretation and difficulties in the standardization of analyses. To characterize high-dimensional cytometric data, we demonstrate the use of probability state modeling (PSM) to visualize the differentiation of effector/memory CD8(+) T cells. With this model, four major CD8(+) T-cell subsets can be easily identified using the combination of three markers, CD45RA, CCR7 (CD197), and CD28, with the selection markers CD3, CD4, CD8, and side scatter (SSC). PSM enables the translation of complex multicolor flow cytometric data to pathway-specific cell subtypes, the capability of developing averaged models of healthy donor populations, and the analysis of phenotypic heterogeneity. In this report we also illustrate the heterogeneity in memory T-cell subpopulations as branched differentiation markers that include CD127, CD62L, CD27, and CD57.
Full-text · Article · Aug 2013 · Journal of immunological methods
[Show abstract][Hide abstract] ABSTRACT: Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.
[Show abstract][Hide abstract] ABSTRACT: A variety of RNA analysis technologies are available for the detection of RNA transcripts from bulk cell populations. However, the techniques for RNA detection from individual cells have been limited. Here we adapt a novel in situ signal amplification method (the RNAScope® detection platform) for the analysis of intracellular RNAs in individual cells by flow cytometry. Using novel target-specific probes that were designed to suppress background signals, we demonstrate the specific detection of HIV gag RNAs in HIV-infected cellular samples, in addition to bcr and abl mRNAs in the K562 cell line. This method was capable of distinguishing cells expressing low abundance RNA transcripts and correlated well with quantitative imaging analysis. Furthermore, multiple distinct RNA targets were simultaneously detected with a high specificity without interference. Overall, the sensitivity and specificity of this method will be useful for the analysis of functionally important RNA species from individual cells, even at very low copy numbers.
[Show abstract][Hide abstract] ABSTRACT: We aim to characterize VTX-2337, a novel Toll-like receptor (TLR) 8 agonist in clinical development, and investigate its potential to improve monoclonal antibody-based immunotherapy that includes the activation of natural killer (NK) cells.
HEK-TLR transfectants were used to compare the selectivity and potency of VTX-2337, imiquimod, CpG ODN2006, and CL075. The ability of VTX-2337 to induce cytokine and chemokine production from human peripheral blood mononuclear cells (PBMC) and activation of specific immune cell subsets was examined. The potential for VTX-2337 to activate NK cell activity through direct and indirect mechanisms was also investigated. Finally, we tested the potential for VTX-2337 to augment antibody-dependent cell-mediated cytotoxicity (ADCC), especially in individuals with low-affinity FcγR3A single-nucleotide polymorphism (SNP).
VTX-2337 selectively activates TLR8 with an EC(50) of about 100 nmol/L and stimulates production of TNFα and interleukin (IL)-12 from monocytes and myeloid dendritic cells (mDC). VTX-2337 stimulates IFNγ production from NK cells and increases the cytotoxicity of NK cells against K562 and ADCC by rituximab and trastuzumab. Effects of VTX-2337 on NK cells were, in part, from direct activation as increased IFNγ production and cytotoxic activity were seen with purified NK cells. Finally, VTX-2337 augments ADCC by rituximab in PBMCs with different FcγR3A genotypes (V/V, V/F, and F/F at position 158).
VTX-2337 is a novel small-molecule TLR8 agonist that activates monocytes, DCs, and NK cells. Through the activation of NK cells, it has the potential to augment the effectiveness of monoclonal antibody treatments where a polymorphism in FcγR3A limits clinical efficacy.
Full-text · Article · Nov 2011 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: To facilitate development of innovative immunotherapy approaches, especially for treatment concepts exploiting the potential benefits of personalized therapy, there is a need to develop and validate tools to identify patients who can benefit from immunotherapy. Despite substantial effort, we do not yet know which parameters of antitumor immunity to measure and which assays are optimal for those measurements.
The iSBTc-SITC (International Society for Biological Therapy of Cancer-Society for Immunotherapy of Cancer), FDA (Food and Drug Administration), and NCI (National Cancer Institute) partnered to address these issues for immunotherapy of cancer. Here, we review the major challenges, give examples of approaches and solutions, and present our recommendations.
Although specific immune parameters and assays are not yet validated, we recommend following standardized (accurate, precise, and reproducible) protocols and use of functional assays for the primary immunologic readouts of a trial; consideration of central laboratories for immune monitoring of large, multi-institutional trials; and standardized testing of several phenotypic and functional potential potency assays specific to any cellular product. When reporting results, the full QA (quality assessment)/QC (quality control) should be conducted and selected examples of truly representative raw data and assay performance characteristics should be included. Finally, to promote broader analysis of multiple aspects of immunity, and gather data on variability, we recommend that in addition to cells and serum, RNA and DNA samples be banked (under standardized conditions) for later testing. We also recommend that sufficient blood be drawn to allow for planned testing of the primary hypothesis being addressed in the trial, and that additional baseline and posttreatment blood is banked for testing novel hypotheses (or generating new hypotheses) that arise in the field.
Full-text · Article · May 2011 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: When evaluating candidate prophylactic HIV and cancer vaccines, intracellular cytokine staining (ICS) assays that measure the frequency and magnitude of antigen-specific T-cell subsets are one tool to monitor immunogen performance and make product advancement decisions. To assess the inter-laboratory assay variation among multiple laboratories testing vaccine candidates, the NIH/NIAID/DAIDS in collaboration with BD Biosciences implemented an ICS Quality Assurance Program (QAP). Seven rounds of testing have been conducted in which 16 laboratories worldwide participated. In each round, IFN-γ, IL-2 and/or TNF-α responses in CD4+ and CD8+ T-cells to CEF or CMV pp65 peptide mixes were tested using cryopreserved peripheral blood mononuclear cells (PBMC) from CMV seropositive donors. We found that for responses measured above 0.2%, inter-laboratory %CVs were, on average, 35%. No differences in inter-laboratory variation were observed if a 4-color antibody cocktail or a 7-color combination was used. Moreover, the data allowed identification of important sources of variability for flow cytometry-based assays, including: number of collected events, gating strategy and instrument setup and performance. As a consequence, in this multi-site study we were able to define pass and fail criteria for ICS assays, which will be adopted in the subsequent rounds of testing and could be easily extrapolated to QAP for other flow cytometry-based assays.
Preview · Article · Jan 2011 · Journal of immunological methods
[Show abstract][Hide abstract] ABSTRACT: In recent years, techniques that combine the use of phospho-specific antibodies and multiparameter flow cytometry have been developed for the detection of protein phosphorylation at the single cell level. Flow cytometry is uniquely suited for this type of analysis, as it can measure functional and phenotypic markers in the context of complex cell populations. Phosphorylation can be assessed simultaneously in multiple cell subsets, and due to the small sample sizes required, and the rapid analyses of large numbers of cells in this approach, rare cell analysis is possible without the ex vivo expansion of cells.In this chapter, we detail flow cytometric protocols for the detection of intracellular phospho-proteins in samples derived from whole blood and peripheral blood mononuclear cell preparations. These protocols define steps for cell activation, fixation, permeabilization, and staining by phospho-specific and phenotyping antibodies. We discuss technical difficulties inherent to this technique and suggest solutions to commonly encountered problems. Additionally, we show examples of phospho-protein detection in lymphocyte subsets, dendritic cells, and monocytes activated with various stimuli, including mitogens, cytokines, and superantigens. Finally, we highlight a potential clinical trial application for this flow cytometric assay as a platform for pharmacodynamic monitoring of kinase inhibitors.
No preview · Article · Jan 2011 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: The extraordinarily high level of genetic variation of HIV-1 env genes poses a challenge to obtain antibodies that cross-react with multiple subtype Env glycoproteins. To determine if cross-reactive monoclonal antibodies (mAbs) to highly conserved epitopes in HIV-1 envelope glycoproteins can be induced, we immunized mice with wild-type or consensus HIV-1 Env proteins and characterized a panel of ten mAbs that reacted with varying breadth to subtypes A, B, C, D, F, G, CRF01_AE, and a highly divergent SIVcpzUS Env proteins by ELISA and Western blot analysis. Two mAbs (3B3 and 16H3) cross-reacted with all tested Env proteins, including SIVcpzUS Env. Surface plasmon resonance analyses showed both 3B3 and 16H3 bound Env proteins with high affinity. However, neither neutralized primary HIV-1 pseudoviruses. These data indicate that broadly reactive non-neutralizing monoclonal antibodies can be elicited, but that the conserved epitopes that they recognize are not present on functional virion trimers. Nonetheless, such mAbs represent valuable reagents to study the biochemistry and structural biology of Env protein oligomers.
[Show abstract][Hide abstract] ABSTRACT: NK cells represent a critical component of the host innate immune response to viral infection and tumor transformation. Nevertheless, the fate of recently degranulated NK cells subsequent to a primary target cell interaction remains largely unexplored. Here, we investigated the long-term viability and killing potential of human NK cells following target cell lysis using live-sorting of CD107a-degranulated NK cells. We observed that sorted CD107a+ NK cells exhibited continued lytic potential against a wide variety of target cells, including tumor and virally infected target cells. CD107a-positive- and CD107a-negative-sorted NK cells displayed similar long-term viability, killing potential, and response to inflammatory cytokines such as IL-2, IL-15, and IFN-alpha. Interestingly, we observed that the CD107a signature is remarkably stable over time and that recently degranulated NK cells exhibit an amplification of CD107 expression immediately following a target cell interaction. Together, our data expand previous data showing that NK cells retain the capacity to kill multiple target cells in succession and reveal that NK viability, cytotoxicity, and response to inflammatory cytokines are not altered following a primary target cell interaction. Overall, our data argue for the strength of the NK cell compartment in the continuous surveillance of tumor and virally infected cells in the body and highlight the use of using CD107a expression as a stable marker for NK cytotoxicity.
Full-text · Article · Mar 2009 · Journal of leukocyte biology
[Show abstract][Hide abstract] ABSTRACT: Intracellular cytokine staining (ICS) is a common method for rapid quantitation of cytokine-producing antigen-specific T cells. T cell production of IFNgamma in particular, and more recently IL-2 as well, is often taken as a measure of vaccine immunogenicity in experimental vaccine trials. As more fluorochromes become available for use in ICS and other applications detecting intracellular markers, the selection of optimal fluorochrome combinations becomes correspondingly more complicated. Additionally, as more sophisticated flow cytometers become available, more attention is being paid to potential result variability from one instrument to another. This review summarizes an oral presentation given at MASIR 2008, January 30-Feb 1, 2008, in La Plagne, France. We focus on issues associated with multiparameter (>four color) flow cytometry, including matching antibody specificities with available fluorochromes and techniques to optimize fluorochrome combinations. We examine issues specific to intracellular staining as well as broader topics such as instrument setup, experimental controls, sample management, and analysis of multiparameter data sets. Particular emphasis is placed on the use of lyophilized cells, antibodies, beads, peptides, etc. (collectively known as "lyoplates"), which can decrease experiment-to-experiment variability as well as processing time. Most clinical trials compile results from multiple testing sites, using data that was acquired on-site in each location. We present data from two different ongoing multi-laboratory standardization studies, one involving 15 laboratories and one involving nine. We identify issues of variability and, where possible, offer solutions.
[Show abstract][Hide abstract] ABSTRACT: Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNgamma-based ELISPOT and cytokine flow cytometry (CFC), as well as tetramer assays.
Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen.
These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.
[Show abstract][Hide abstract] ABSTRACT: Primary simian immunodeficiency virus (SIV) infections of rhesus macaques result in the dramatic depletion of CD4(+) CCR5(+) effector-memory T (T(EM)) cells from extra-lymphoid effector sites, but in most infections, an increased rate of CD4(+) memory T cell proliferation appears to prevent collapse of effector site CD4(+) T(EM) cell populations and acute-phase AIDS. Eventually, persistent SIV replication results in chronic-phase AIDS, but the responsible mechanisms remain controversial. Here, we demonstrate that in the chronic phase of progressive SIV infection, effector site CD4(+) T(EM) cell populations manifest a slow, continuous decline, and that the degree of this depletion remains a highly significant correlate of late-onset AIDS. We further show that due to persistent immune activation, effector site CD4(+) T(EM) cells are predominantly short-lived, and that their homeostasis is strikingly dependent on the production of new CD4(+) T(EM) cells from central-memory T (T(CM)) cell precursors. The instability of effector site CD4(+) T(EM) cell populations over time was not explained by increasing destruction of these cells, but rather was attributable to progressive reduction in their production, secondary to decreasing numbers of CCR5(-) CD4(+) T(CM) cells. These data suggest that although CD4(+) T(EM) cell depletion is a proximate mechanism of immunodeficiency, the tempo of this depletion and the timing of disease onset are largely determined by destruction, failing production, and gradual decline of CD4(+) T(CM) cells.
Full-text · Article · Oct 2007 · Journal of Experimental Medicine
[Show abstract][Hide abstract] ABSTRACT: The overall prevalence with which endogenous tumor Ags induce host T cell responses is unclear. Even when such responses are detected, they do not usually result in spontaneous remission of the cancer. We hypothesized that this might be associated with a predominant phenotype and/or cytokine profile of tumor-specific responses that is different from protective T cell responses to other chronic Ags, such as CMV. We detected significant T cell responses to CEA, HER-2/neu, and/or MAGE-A3 in 17 of 21 breast cancer patients naive to immunotherapy. The pattern of T cell cytokines produced in response to tumor-associated Ags (TAAs) in breast cancer patients was significantly different from that produced in response to CMV or influenza in the same patients. Specifically, there was a higher proportion of IL-2-producing CD8(+) T cells, and a lower proportion of IFN-gamma-producing CD4(+) and/or CD8(+) T cells responding to TAAs compared with CMV or influenza Ags. Finally, the phenotype of TAA-responsive CD8(+) T cells in breast cancer patients was almost completely CD28(+)CD45RA(-) (memory phenotype). CMV-responsive CD8(+) T cells in the same patients were broadly distributed among phenotypes, and contained a high proportion of terminal effector cells (CD27(-)CD28(-)CD45RA(+)) that were absent in the TAA responses. Taken together, these results suggest that TAA-responsive T cells are induced in breast cancer patients, but those T cells are phenotypically and functionally different from CMV- or influenza-responsive T cells. Immunotherapies directed against TAAs may need to alter these T cell signatures to be effective.
Full-text · Article · Sep 2007 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: In vivo, several mechanisms have been postulated to protect HIV-1-infected cells from NK surveillance. In vitro, previous research indicates HIV-1-infected autologous CD4(+) primary T cells are resistant to NK lysis. We hypothesized that NK lysis of HIV-1-infected target cells would be augmented by the presence of accessory cells and/or accessory cell factors. In this study, we show that stimulation of plasmacytoid dendritic cells (PDC) with the TLR9 agonist, CpG ODN 2216, triggered NK lysis of HIV-1-infected autologous CD4(+) primary T cells. PDC-stimulated NK lysis was dependent upon MHC class I (MHC-I) down-regulation on infected cells, and primary HIV-1 isolates that exhibited enhanced MHC-I down-regulation were more susceptible to NK-mediated lysis. PDC-stimulated NK lysis of HIV-1-infected autologous CD4(+) primary T cells was blocked by neutralizing Abs to type 1 IFN and was perforin/granzyme dependent. Overall, our data suggest that HIV-infected cells are not innately resistant to NK lysis, and that exogenous NK stimulation derived from PDC can trigger NK cytotoxicity against HIV-1-infected autologous CD4(+) primary T cells.
Full-text · Article · Sep 2007 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Monocyte-derived-dendritic-cells (MDDC) are the major DC type used in vaccine-based clinical studies for a variety of cancers. In order to assess whether in vitro differentiated MDDC from cryopreserved PBMC of cancer patients are functionally distinct from those of healthy donors, we compared these cells for their expression of co-stimulatory and functional markers. In addition, the effect of cryopreservation of PBMC precursors on the quality of MDDC was also evaluated using samples from healthy donors.
Using flow cytometry, we compared normal donors and cancer patients MDDC grown in the presence of GM-CSF+IL-4 (immature MDDC), and GM-CSF+IL-4+TNFalpha+IL-1beta+IL-6+PGE-2 (mature MDDC) for (a) surface phenotype such as CD209, CD83 and CD86, (b) intracellular functional markers such as IL-12 and cyclooxygenase-2 (COX-2), (c) ability to secrete IL-8 and IL-12, and (d) ability to stimulate allogeneic and antigen-specific autologous T cells.
Cryopreservation of precursors did affect MDDC marker expression, however, only two markers, CD86 and COX-2, were significantly affected. Mature MDDC from healthy donors and cancer patients up-regulated the expression of CD83, CD86, frequencies of IL-12+ and COX-2+ cells, and secretion of IL-8; and down-regulated CD209 expression relative to their immature counterparts. Compared to healthy donors, mature MDDC generated from cancer patients were equivalent in the expression of nearly all the markers studied and importantly, were equivalent in their ability to stimulate allogeneic and antigen-specific T cells in vitro.
Our data show that cryopreservation of DC precursors does not significantly affect the majority of the MDDC markers, although the trends are towards reduced expression of co-stimulatory makers and cytokines. In addition, monocytes from cryopreserved PBMC of cancer patients can be fully differentiated into mature DC with phenotype and function equivalent to those derived from healthy donors.
Preview · Article · Feb 2007 · Journal of Immune Based Therapies and Vaccines
[Show abstract][Hide abstract] ABSTRACT: The kinetics of recovery for innate immune effectors following antiretroviral therapy are unknown.
Multiple sequential cryopreserved samples (viremic and ART-suppressed) from 66 patients enrolled in the Women's Interagency HIV Study or Multicenter AIDS Cohort Study cohorts (median follow-up, 700 days) were analyzed to determine natural killer, dendritic and T-cell changes by flow cytometry. Functional parameters were also measured in a subset of samples. Changes over time were analyzed by mixed-effect modeling based on a linear spline with a single knot at 270 days.
Following viral suppression, a rapid rise in CD4 and white blood cell counts and a decline in T-cell activation were confirmed. However, natural killer cell subsets increased after 270 days of therapy, with a negative effect by baseline CD4%. CD123+ plasmacytoid but not myeloid dendritic cells showed a trend to increase during the first 270 days with a positive effect of baseline CD4%; plasmacytoid dendritic cell-induced interferon-alpha production significantly increased by end of follow-up.
The kinetics of natural killer and plasmacytoid dendritic cell recovery are markedly different from those of T-cell subsets, indicative of early and delayed benefits of suppressive regimens.