Mary Beth Hanley - Academia.edu (original) (raw)

Papers by Mary Beth Hanley

Blood, 2019

Chimeric antigen receptor T-cells (CAR-T) have emerged as an extremely promising therapy for hema... more Chimeric antigen receptor T-cells (CAR-T) have emerged as an extremely promising therapy for hematological malignancies. The immunophenotype of apheresis material and the CAR-T cell product is known to be predictive of the likelihood of response to treatment of certain malignancies. Central memory and stem cell-like memory T cell phenotypes are associated with a more sustained proliferative response and long-term CAR-T persistence (Fraietta et al, Nature Medicine, 2018). There is an unmet need for standardized methods and reagents to reliably profile the memory phenotype of CAR-Ts to better evaluate product quality, and support improvements in CAR-T manufacturing. The BD Biosciences dried memory T-cell panel contains a pre-validated mixture of 7 antibodies for the identification of naïve, stem cell memory, central memory and effector memory CD4+ and CD8+ T cell subsets. The pre-mixed dried antibody tube offers consistency in staining profiles over time and reduces the risk of operat...

The Journal of Infectious Diseases, Feb 1, 2002

T cell dynamics were studied in human immunodeficiency virus-infected patients who continued usin... more T cell dynamics were studied in human immunodeficiency virus-infected patients who continued using antiretroviral therapy despite detectable plasma viremia (RNA copies >2500 /mL). CD4 + cell fractional replacement rates, measured by the deuterated glucose technique, were lower in treated patients with detectable viremia than in untreated patients and were similar to those in patients with undetectable viremia. Cell cycle and activation markers exhibited similar trends. For any level of viremia, CD4 + cell fractional replacement rates were lower in patients with drug-resistant virus than in patients with wild-type virus, which suggests that the resistant variant was less virulent. Interruption of treatment in patients with drug-resistant viremia resulted in increased CD4 + cell activation, increased CD4 + cell turnover, and decreased CD4 + cell counts. These data indicate that partial virus suppression reduces CD4 + cell turnover and activation, thereby resulting in sustained CD4 + cell gains, and that measurements of T cell dynamics may provide an in vivo marker of viral virulence.

Journal of Immunology, Jul 15, 2003

Superantigen-mediated deletion of speci c T cell receptor V beta subsets in the SCID-hu Thy/Liv m... more Superantigen-mediated deletion of speci c T cell receptor V beta subsets in the SCID-hu Thy/Liv mouse is induced by staphylococcal enterotoxin B, but not HIV-1.

Nature Medicine, 1999

The dynamic basis for T-cell depletion in late-stage HIV-1 disease remains controversial. Using a... more The dynamic basis for T-cell depletion in late-stage HIV-1 disease remains controversial. Using a new, non-radioactive, endogenous labeling technique, we report direct measurements of circulating T-cell kinetics in normal and in HIV-1-infected humans. In healthy, HIV-1-seronegative subjects, CD4+ and CD8+ T cells had half-lives of 87 days and 77 days, respectively, with absolute production rates of 10 CD4+ T cells/microl per day and 6 CD8+ T cells/microl per day. In untreated HIV-1-infected subjects (with a mean CD4 level of 342 cells/microl), the half-life of each subpopulation was less than 1/3 as long as those of healthy, HIV-1-seronegative subjects but was not compensated by an increased absolute production rate of CD4+ T cells. After viral replication was suppressed by highly active antiretroviral therapy for 12 weeks, the production rates of circulating CD4+ and CD8+ T cells were considerably elevated; the kinetic basis of increased CD4 levels was greater production, not a longer half-life, of circulating cells. These direct measurements indicate that CD4+ T-cell lymphopenia is due to both a shortened survival time and a failure to increase the production of circulating CD4+ T cells. Our results focus attention on T-cell production systems in the pathogenesis of HIV-1 disease and the response to antiretroviral therapy.

Journal of Clinical Investigation, Mar 1, 2000

Journal of Immunological Methods, 2011

When evaluating candidate prophylactic HIV and cancer vaccines, intracellular cytokine staining (... more 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 were 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.

Journal of Clinical Investigation, Sep 15, 2003

Antigenic stimulation of T cells gives rise to short-lived effector cells and long-lived memory c... more Antigenic stimulation of T cells gives rise to short-lived effector cells and long-lived memory cells. We used two stable isotope-labeling techniques to identify kinetically distinct subpopulations of T cells and to determine the effect of advanced infection with HIV-1. Long-term deuterated water (2 H 2 O) incorporation into DNA demonstrated biphasic accrual of total and of memory/effector (m/e)-phenotype but not naive-phenotype T cells, consistent with the presence of short-lived and longer-lived subpopulations within the m/e-phenotype T cell pool. These results were mirrored by biphasic die-away kinetics in m/e-but not naive-phenotype T cells after short-term 2 H-glucose labeling. Persistent label retention was observed in a subset of m/e-phenotype T cells (presumably memory T cells), confirming the presence of T cells with very different life spans in humans. In advanced HIV-1 infection, much higher proportions of T cells were short-lived, compared to healthy controls. Effective long-term anti-retroviral therapy restored values to normal. These results provide the first quantitative evidence that long-lived and quiescent T cells do indeed predominate in the T cell pool in humans and determine T cell pool size, as in rodents. The greatest impact of advanced HIV-1 infection is to reduce the generation of long-lived, potential progenitor T cells.

Journal of Immunology, May 1, 2013

A variety of RNA analysis technologies is available for the detection of RNA transcripts from bul... more A variety of RNA analysis technologies is available for the detection of RNA transcripts from bulk cell populations. However, the techniques for RNA detection from single cells have been limited due to lack of sensitivity in detecting specific mRNAs present at low abundance in individual cells. By designing novel target-specific probes and adapting an in situ signal amplification method based on the RNAscope® detection platform, we demonstrate the specific and sensitive detection of intracellular RNAs in single cells by flow cytometry (RNAFlow). This method had sufficient sensitivity to distinguish cells containing low abundance RNA transcript. Furthermore, multiple distinct RNA targets were simultaneously detected with a high specificity in single cells without interference. The method can quantify the frequency of cells expressing specific RNA as well as the number of RNA copies in each expression-positive cell. We will present quantitative data to characterize RNA target-specific signal detection, demonstrated in applications such as HIV infection and immune activation. The RNAFlow assay, independently or as combined assay with co-protein detection, represents a valuable research tool for the specific and sensitive detection of multiple RNA transcripts in individual cells in heterogeneous biological specimens. Overall, this method will be useful for the analysis of functionally important RNA species from single cells, even at very low copy numbers.

The Journal of Immunology

(a) A pseudocolor merged segmentation mask image obtained using Cell ... more (a) A pseudocolor merged segmentation mask image obtained using Cell Profiler software analysis of a mixture of H9 and H9IIIB cells (left image); HIV gag-Alexa Fluor® 647, 18 s-FITC, and DAPI counterstain. The calculated frequency of HIV gag+ cells (within the 18 s+DAPI+ cells) is shown on the image. The RNA flow cytometry plots of the same mixture of H9 and H9IIIB cells. (b) Flow cytometry overlay histograms of HIV gag RNA in HIV-infected PBMCs (solid line) and mock-infected PBMCs (tinted with dashed line) in freshly acquired PBMCs (left plot) and the same PBMCs after cryopreservation (right plot).</p

(a) Representative color merged 60x images of the HIV+ cell line H9II... more (a) Representative color merged 60x images of the HIV+ cell line H9IIIB (left) and HIV negative cell line H9 (right); HIV gag RNA-Alexa Fluor® 647 (green), 18 s rRNA-FITC (red), and Nuclei-DAPI (blue). (b) The frequency of HIV gag RNA-positive cells within the 18 s+DAPI+ population was calculated after spiking HIV+ cells into the negative cell population at the stated percentages in the table. The image below the table is representative of data from image segmentation analysis. (c) 40x image of CD4 immunophenotyping overlaid with HIV gag RNA in HIV-infected PBMCs; Anti-CD4 antibody-Alexa Fluor® 488 (red), HIV gag RNA-Alexa Fluor® 546 (green), and DAPI (blue).</p

(a) Breakdown of spot numbers from image analysis (bcr+18 s+DAPI+) in... more (a) Breakdown of spot numbers from image analysis (bcr+18 s+DAPI+) into bins (x-axis) with corresponding frequencies (y-axis). (b) RNA flow histogram of K562 cells split into groups using the percentages obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057002#pone-0057002-g005" target="_blank">Figure 5A</a> with the corresponding MFIs (x-axis and table). Colors correspond to the spot bin numbers in the bar chart in 5a. The lowest bin (spot count 1–5) was further subdivided and is shown in the inset on the histogram with the corresponding spot count on each peak. (c) Comparison chart of mean integrated intensities (black bars) obtained from image analysis and MFIs (white bars) obtained from RNA flow cytometry analysis (5b). Standard deviations are shown for each group. The coefficient of determination (r2) is displayed on the graph (0.988). (d) Spot intensities for each spot bin with correlating standard deviations for each group. The lower bin (1–5) was further subdivided and is shown on the left side of the chart. (e) Histogram showing sort gates for the different bcr MFI subsets in the K562 cell line. Outline overlay images shown to the right of the histogram are representative of the sorted cells from each subset sorted showing bcr RNA (green), 18 s rRNA (red), and DAPI counterstaining (blue). The bcr spot count average per cell is listed below its respective image.</p

(a) Pseudocolor-merged 40x images of a 1:1 mixture of H9 and H9IIIB c... more (a) Pseudocolor-merged 40x images of a 1:1 mixture of H9 and H9IIIB cells via the slide-based RNA detection method (left) and the suspension method (right) of HIV gag RNA-Alexa Fluor® 546, 18 s rRNA-FITC, and DAPI. The calculated frequencies for HIV gag+18 s+ RNA for each are depicted in each image. (b) Mean intensity comparison of HIV gag for different HIV gag spot count ranges (bins) with the suspension-based and slide-based methods. Error bars depict the standard deviation within each spot count bin. The correlation coefficient, r, and the coefficient of determination, r2, are shown on the graph.</p

(a) Flow cytometry plots from three-probe (bcr, abl, 18 s) RNA analys... more (a) Flow cytometry plots from three-probe (bcr, abl, 18 s) RNA analysis in the K562 cell line: 18 s rRNA (left) and bcr vs. abl RNA in the 18 s+ events (right). (b) 60x pseudocolored images of sorted bcr+abl+18 s+ cells from K562 cells; Alexa Fluor® 647 labeled bcr (red), Alexa Fluor® 546 labeled abl (green). The bcr/abl fusion transcripts are shown in yellow due to the merging of both the Alexa Fluor® 546 and Alexa Fluor® 647 dyes. Cells were counterstained with DAPI. (c) Graph showing the effects of RNA staining by multiple probes (bcr+abl+18 s) compared to a single target probe on MFIs in RNA flow cytometry. Bars represent the standard deviation for duplicate samples run.</p

Diversity is a hallmark of the immune system. The repertoires of B cells and of CD4 and CD8 T cel... more Diversity is a hallmark of the immune system. The repertoires of B cells and of CD4 and CD8 T cells each consist of more than 108 different clonotypes each characterized by a unique receptor Qi et al. (2014); Goncalves et al. (2017). Each immune response is characterized by a large panel of different cytokines with –partly overlapping– functions. Each individual is characterized by a unique combination of MHC molecules that play an essential role in the selection of peptides presented to the cellular immune system. MHC loci are the most polymorphic genes known for vertebrates, i.e., for most loci several hundreds of alleles have been identified within each population. However, each individual inherits only a limited number of MHC genes from its parents, and expresses about 10 different MHC molecules. We will here address the evolutionary questions why lymphocytes are so diverse within an individual, and why MHC molecules are diverse at the population level, and not diverse within an...

Nature medicine, 1999

Cytometry Part A, 2014

The application of fluorescently-labeled antibodies for flow cytometric identification and charac... more The application of fluorescently-labeled antibodies for flow cytometric identification and characterization of specific cell types within heterogeneous populations by their protein expression profile is well established. While detection of proteins is informative, concomitant transcript analysis in the same cells would provide a more complete and comprehensive view of intracellular signaling events. We recently reported on the efficient detection of RNA in suspension cells for flow cytometric analysis. The improved RNA flow cytometry procedure described here allows for the specific labeling of multiple RNA species, and is compatible with antibody-based targeting of extracellular and intracellular antigens for multiplexing purposes. To show proof of concept, human peripheral blood mononuclear cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin for a maximum of 5 h, during which their CD4 and interferongamma (IFN-c) transcript and protein levels were monitored. Substantial and increasing numbers of IFN-c mRNA 1 cells were detected within 30 min after initiation of induction, while IFN-c protein 1 cells could only be discerned at 1 h and beyond. Surprisingly, resting lymphocytes contained less CD4 mRNA but more of the protein per cell compared with monocytes, revealing a difference in the relationship of transcript and protein levels in these two cell types. We additionally applied monensin, which is commonly used to block cytokine secretion, and found that IFN-c mRNA can still be analyzed consistently using the improved RNA flow cytometry staining method. Notably, a subset of IFN-c mRNA-/protein 1 cells that were not observed in the absence of monensin became apparent at the 5-h mark. This subset probably represents cells that have accumulated IFN-c protein, but no longer transcribe mRNA. Collectively, the results described here exemplify how the improved RNA flow cytometry labeling procedure can be applied to simultaneously assess mRNA and protein dynamics to gain insight into the regulation of gene transcription and translation in individual cells. V

STEM CELLS, 2005

Growth hormone (GH) has been shown to have significant positive effects on hemato-lymphopoiesis i... more Growth hormone (GH) has been shown to have significant positive effects on hemato-lymphopoiesis in rodent models and, more recently, to increase thymic mass and circulating naïve CD4 + T cells in humans infected with the human immunodeficiency virus, type 1. To determine whether the latter effects on human T lymphopoiesis might be due, at least in part, to effects on the bone marrow (BM), we examined the specific effects of GH and its proximal mediator, insulin-like growth factor I (IGF-I), on human multilineage hematopoiesis in fetal BM (FBM). Using in vitro analysis, we found that GH and IGF-I each stimulated the expansion of primitive multilineage CD34 + CD38 − hematopoietic progenitor cells and increased yields of several hematopoietic subpopulations, including CD34 + CD38 + CD10 + lymphoid progenitor cells. Additionally, GH and IGF-I had direct effects on FBM stromal elements,inducing the expansion of myeloid-like CD45 + CD14 + FBM stromal cells and enhancing production of the hematopoietic cytokine interleukin-3 by fibroblast-like CD45 − CD10 + FBM stromal cells. Surface expression of GH and type-I IGF receptors correlated with the observed biologic responses to these hormones. Whereas GH enhanced the proliferation of FBM progenitors and stroma, IGF-I exerted a predominantly antiapoptotic effect. Finally, both GH and IGF-I stimulated the generation of hematopoietic colony forming cells. These findings identify specific targets of GH and IGF-I within human FBM, and demonstrate direct and indirect effects that may contribute to GH-mediated enhancement of human hemato-lymphopoiesis.

The Journal of Infectious Diseases, 2002

The Journal of Immunology, 2003

IL-7 is a critical component of thymopoiesis in animals and has recently been shown to play an im... more IL-7 is a critical component of thymopoiesis in animals and has recently been shown to play an important role in T cell homeostasis. Although there is increasing interest in the use of IL-7 for the treatment of lymphopenia caused by the HIV type 1, evidence that IL-7 may accelerate HIV replication has raised concerns regarding its use in this setting. We sought to identify the effects of IL-7 on human thymocyte survival and to determine the impact of IL-7 administration on in vivo HIV infection of the human thymus. Using in vitro analysis, we show that IL-7 provides potent anti-apoptotic and proliferative signals to early thymocyte progenitors. Analysis of CD34+ subpopulations demonstrates that surface IL-7 receptor is expressed on most CD34highCD5+CD1a− thymocytes and that this subpopulation appears to be one of the earliest maturation stages responsive to the effects of IL-7. Thus, IL-7 provides survival signals to human thymocytes before surface expression of CD1a. CD4+CD8+ thymo...