Beverly Z. Packard

Lytic Granule Loading of CD8+ T Cells Is Required for HIV-Infected Cell Elimination Associated with Immune Control

Virus-specific CD8+ T cells probably mediate control over HIV replication in rare individuals, termed long-term nonprogressors (LTNPs) or elite controllers. Despite extensive investigation, the mechanisms responsible for this control remain incompletely understood. We observed that HIV-specific CD8+ T cells of LTNPs persisted at higher frequencies than those of treated progressors with equally low amounts of HIV. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This function required lytic granule loading of effectors and delivery of granzyme B to target cells. Defective cytotoxicity of progressor effectors could be restored after treatment with phorbol ester and calcium ionophore. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. They also demonstrate that lytic granule contents of memory cells are a critical determinant of cytotoxicity that must be induced for maximal per-cell killing capacity.

Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates

We have developed a non-radioactive flow-cytometry assay to monitor and quantify the target-cell killing activities mediated by cytotoxic T lymphocytes (CTLs). This flow-cytometry CTL (FCC) assay is predicated on measurement of CTL-induced caspase activation in target cells through detection of the specific cleavage of fluorogenic caspase substrates. Here we show that this assay reliably detects antigen-specific CTL killing of target cells, and demonstrate that it provides a more sensitive, more informative and safer alternative to the standard 51Cr-release assay most often used to quantify CTL responses. The FCC assay can be used to study CTL-mediated killing of primary host target cells of different cell lineages, and enables the study of antigen-specific cellular immune responses in real time at the single-cell level. As such, the FCC assay can provide a valuable tool for studies of infectious disease pathogenesis and development of new vaccines and immunotherapies.

High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses.

We have developed a high‐throughput platform to detect the presence of HIV‐1 and SIV‐specific ADCC‐mediating antibody responses. The assay is based on the hydrolysis of a cell‐permeable fluorogenic peptide substrate containing a sequence recognized by the serine protease, Granzyme B (GzB). GzB is delivered into target cells by cytotoxic effector cells as a result of antigen (Ag)‐specific Ab‐Fcγ receptor interactions. Within the target cells, effector cell‐derived GzB hydrolyzes the substrate, generating a fluorescent signal that allows individual target cells that have received a lethal hit to be identified by flow cytometry. Results are reported as the percentage of target cells with GzB activity (%GzB). Freshly isolated or cryopreserved PBMC and/or NK cells can be used as effector cells. CEM.NKR cells expressing the CCR5 co‐receptor are used as a target cells following: (i) coating with recombinant envelope glycoprotein, (ii) infection with infectious molecular clones expressing the Env antigens of primary and lab adapted viruses, or (iii) chronic infection with a variant of HIV‐1/IIIB, termed A1953. In addition, primary CD4+ T cells infected with HIV‐1 in vitro can also be used as targets. The assay is highly reproducible with a coefficient of variation of less than 25%. Target and effector cell populations, in the absence of serum/plasma, were used to calculate background (8.6 ± 2.3%). We determined that an initial dilution of 1:50 and 1:100 is required for testing of human and non‐human primate samples, respectively. This assay allows for rapid quantification of HIV‐1 or SIV‐specific ADCC‐mediating antibodies that develop in response to vaccination, or in the natural course of infection, thus providing researchers with a new methodology for investigating the role of ADCC‐mediating antibodies as correlates of control or prevention of HIV‐1 and SIV infection.

Direct visualization of protease activity on cells migrating in three-dimensions

Determining the specific role(s) of proteases in cell migration and invasion will require high-resolution imaging of sites of protease activity during live-cell migration through extracellular matrices. We have designed a novel fluorescent biosensor to detect localized extracellular sites of protease activity and to test requirements for matrix metalloprotease (MMP) function as cells migrate and invade three-dimensional collagen matrices. This probe fluoresces after cleavage of a peptide site present in interstitial collagen by a variety of proteases including MMP-2, -9, and -14 (MT1-MMP) without requiring transfection or modification of the cells being characterized. Using matrices derivatized with this biosensor, we show that protease activity is localized at the polarized leading edge of migrating tumor cells rather than further back on the cell body. This protease activity is essential for cell migration in native cross-linked but not pepsin-treated collagen matrices. The new type of high-resolution probe described in this study provides site-specific reporting of protease activity and insights into mechanisms by which cells migrate through extracellular matrices; it also helps to clarify discrepancies between previous studies regarding the contributions of proteases to metastasis.

Granzyme B Activity in Target Cells Detects Attack by Cytotoxic Lymphocytes

Lymphocyte-mediated cytotoxicity via granule exocytosis operates by the perforin-mediated transfer of granzymes from CTLs and NK cells into target cells where caspase activation and other death pathways are triggered. Granzyme B (GzB) is a major cytotoxic effector in this pathway, and its fate in target cells has been studied by several groups using immunodetection. In this study, we have used a newly developed cell-permeable fluorogenic GzB substrate to measure this protease activity in three different living targets following contact with cytotoxic effectors. Although no GzB activity is measurable in CTL or NK92 effector cells, this activity rapidly becomes detectable throughout the target cytoplasm after effector-target engagement. We have combined the GzB substrate with a second fluorogenic substrate selective for caspase 3 to allow both flow cytometry and fluorescence confocal microscopy studies of cytotoxicity. With both effectors, caspase 3 activity appears subsequent to that of GzB inside all three targets. Overexpression of Bcl-2 in target cells has minimal effects on lysis, NK- or CTL-delivered GzB activity, or activation of target caspase 3. Detection of target GzB activity followed by caspase 3 activation provides a unique readout of a potentially lethal injury delivered by cytotoxic lymphocytes.

DPSS Yellow-Green 561-nm Lasers for Improved Fluorochrome Detection by Flow Cytometry

Blue‐green 488‐nm laser sources are widespread in flow cytometry but suffer some drawbacks for cell analysis, including their excitation of endogenous proteins (resulting in high cellular autofluorescence) and their less‐than‐optimal coincidence with the excitation maxima of commonly used fluorochromes, including the phycoerythrins (PE). Longer wavelength lasers such as green helium–neons and, more recently, diode‐pumped solid state (DPSS) 532‐nm sources have previously been employed to overcome these difficulties and improve overall sensitivity for PE. In this study, we evaluate an even longer wavelength DPSS 561‐nm for its ability to improve PE and DsRed fluorescent protein detection sensitivity.

Resolution of absorption spectra of rhodamine 6G aggregates in aqueous solution using the law of mass action

Abstract There is striking dissimilarity among the absorption spectra of rhodamine dimers reported by different authors. To address this issue, we measured the absorption spectra of aqueous solutions of rhodamine 6G over a wide range of concentrations. The lack of an isosbestic point near 508 nm indicates the presence of at least three species. Data analysis using the program spectrabind described in [D. Toptygin, L. Brand, Anal. Biochem. 222 (1995) 330] confirms the presence of trimers and tetramers in addition to dimers and monomers. The absorption peaks of the monomer, dimer, trimer, and tetramer are at 526, 498, 485, and 475 nm, respectively.

Hydrophobic adsorption chromatography to reduce nonspecific staining by rhodamine-labeled antibodies

Rhodamine absorbed to protein was removed from rhodamine-conjugated antibody solutions by adsorption to hydrophobic macroporous beads (Bio-Beads SM-2) following gel permeation chromatography. This simple technique eliminated the contaminating free dye more effectively than gel filtration alone, but neither significantly reduced the level of fluorescently conjugated antibody nor altered the latters binding characteristics. Passage of the fluorescent antibody solution over the SM-2 beads eliminated the high background of nonspecific staining caused by internalization of residual free dye.

Caspase 8 Activity in Membrane Blebs After Anti-Fas Ligation

Previous studies of thymocyte apoptosis using a series of cell-permeable fluorogenic peptide substrates showed that Fas cross-linking triggered a caspase cascade in which cleavage of the IETDase (caspase 8-selective) substrate was the earliest caspase activity measured by flow cytometry. This result was expected in light of the abundant evidence for caspase 8 activation as an initiating event in the Fas death pathway. However, when apoptosis was induced by anti-Fas in CTL and the caspase cascade examined by this approach, IETDase activation followed increases in LEHDase, YVHDase, and VEIDase activities (selective for caspases 9, 1, and 6, respectively). When examined by confocal microscopy, anti-Fas-treated CTL showed the early appearance of IETDase-containing plasma membrane vesicles and their release from the CTL surface, followed by activation of other caspase activities in the cell interior. Since these vesicles were not included in the flow cytometry analysis, the early IETDase activity had been underestimated. In contrast to anti-Fas, induction of apoptosis in these CTL by IL-2 withdrawal resulted in early IETDase activity in the cytoplasm, with no plasma membrane vesiculation. Thus, anti-Fas-induced initiation of caspase activity at the plasma membrane may in some cells result in local proteolysis of submembrane proteins, leading to generation of membrane vesicles that are highly enriched in active caspase 8.

Intracellular protease activation in apoptosis and cell- mediated cytotoxicity characterized by cell-permeable fluorogenic protease substrates

Over the past decade the importance of signaling from reporter molecules inside live cells and tissues has been clearly established. Biochemical events related to inflammation, tumor metastasis and proliferation, and viral infectivity and replication are examples of processes being further defined as more molecular tools for live cell measurements become available. Moreover, in addition to quantitating parameters related to physiologic processes, real-time imaging of molecular interactions that compose basic cellular activities are providing insights into understanding disease mechanisms as well as extending clinical efficacy of therapeutic regimens. In this review the use of highly cell-permeable fluorogenic substrates that report protease activities inside live cells is described; applications to defining the molecular events of two cellular processes, i.e., apoptosis and cell-mediated cytotoxicity, are then illustrated.