A fluorescent in situ hybridization method in flow cytometry to detect HIV-1 specific RNA

A fluorescent in situ hybridization method in flow cytometry to detect HIV-1 specific RNA
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  ELSEVIER Journal of Immunological Methods 193 (1996) 167- 176 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ JOURNAL OF IMMUNOLOGICAL METHODS A fluorescent in situ hybridization method in flow cytometry to detect HIV-l specific RNA R.M. Borz’l a, A. Piacentini a, M.C.G. Monaco f, G. Lisignoli a, A. Degrassi d.e, L. Cattini ‘, S. Santi b, A. Facchini a3c* zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR ’ Laboratorio di Imrnunologia e Genetica, Istiruto di Ricercu Codirilla Purti, I.O.R.. Via di Barbiano I / IO. JO136 Bolognn. Ita \ h Luboratorio di Biologiu Cellulnre e Microscopin Elettronica, Istituto di Ricerca Codirilla Purri I.O.R.. Bologna, Italy ’ Isriruto di Clinica Medico e Gastroenterologia. Universitir di Bologna, Bologna, Ita \ d Dipurtirnetzto di Purotogia r Medicinn Sperinzenratr e Clika, Unirersiiir di i/dine, Udine. Italy ’ Consor:io di Ricerche Biomediche. (ink ersith di C/dine. Udirle. Ital> ’ Laborator of Molecrrlar Medicirze and Neuroscience. NINDS. NIH. Bethesda, MD. USA Received 10 May 1995: revised 12 October 1995: accepted 5 February 1996 zyxwvutsrqponmlkjihgfedcbaZYX Abstract In HIV+ patients, the presence of HIV-RNA in plasma and circulating cells has been reported to be a marker of progression but the percentage of transcriptionally active infected cells remains unclear. We have developed a reliable fluorescent in situ hybridization method for the detection of HIV specific RNA by flow cytometry. The procedure was applied to a panel of chronically infected cell lines and to an acutely infected cell line mimicking normal peripheral blood lymphocytes in susceptibility to HIV-l. The cells were fixed in suspension and hybridized by means of an HIV-l genomic probe labeled with digoxigenin-1 I-dUTP. An FITC-labeled anti-digoxigenin antiserum was then applied and the resulting fluorescence signals were analyzed both by flow cytometry and confocal microscopy. Different procedures for double staining HIV-RNA together with virus induced proteins or surface markers were also developed. Plow cytometric detection of in situ hybridization offers the possibility of analyzing thousands of cells in a few seconds and of collecting multiparametric information at the single cell level, thus providing a potential tool for detecting the rare HIV-RNA expressing cells in peripheral blood samples. Keyw0rd.r: Fluorescent in situ hybridization: HIV-RNA; Flow cytometry zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG 1 Introduction information on both the viral life cycle and the In HIV infected cells the progression from latency to the productive phase involves the start of RNA transcription from the integrated proviral template and in situ detection of HIV-RNA provides valuable * Corresponding author. Tel.: + 39-51-6366803: Fax: +39-51- 6366807. magnitude of infection in biological specimens. Al- though HIV mainly infects lymphoid tissues, where the virus is active and progressive even in the clini- tally ‘latent stage’ (Fox et al., 1991; Burke et al., 1993; Embretson et al., 1993a; Pantaleo et al., 1993), blood infection has also recently been recognized to be wider than previously stated. Several authors have reported much higher percentages of peripheral blood 0022.1759/96/ 15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved PII SOO22-1759(96)00070- 1  lymphocytes harboring an integrated provirus (Bagasra et al., 1992; Patterson et al., 1993) and high levels of plasma viremia (representing active viral replication) have been detected in seropositive pa- tients throughout all stages of disease (Piatak et al., 1993). What remains unsettled is the actual percent- age of cells expressing HIV-RNA. The correlation between the number of HIV-RNA producing cells and clinical status was pointed out by Lewis et al. (1990). Utilizing an in situ hybridiza- tion technique and confocal microscopy these au- thors reported a statistically significant difference between symptomatic (AIDS and ARC) and asymp- tomatic patients in terms of mean numbers of HIV- RNA positive cells per 10000 peripheral blood mononuclear cells (PBMCS) and a trend towards an inverse correlation between the percentage of posi- tive PBMCs and the blood count of CD4+ cells. In situ hybridization (ISH) has possibly the high- est sensitivity for the detection and analysis of RNA molecules among conventional (non-amplifying) molecular techniques. its detection limit being lo- 100 molecules per cell (Kawasaki. 1990). The main advantage of this technique is its ability to detect a specific nucleic acid (RNA or DNA) sequence in morphologically intact cells and tissues. This estab- lishes a precise correlation between the target se- quence and the pathological features of the biologi- cal specimen and simultaneously collects different information from the same cell (such as surface markers or activation associated molecules) by means of double staining procedures. A further advantage of ISH techniques is that the hybridized cells can be analyzed by flow cytometry. This is possible by performing ISH on cells fixed in suspension and by using probes either directly or indirectly labeled with a fluorochrome (fluorescent in situ hybridization in flow cytometry or FISH IN FLOW). Flow cytometric analysis offers the possibil- ity of quantifying accurately the FISH signals of thousands of cells in a few seconds and collecting and storing in correlated list modes multiparametric data from a single cell, thus providing a potential tool for the routine analysis of specific nucleic acid sequences in low frequency cell populations. This paper describes the development of a flue- rescent in situ hybridization method to detect HIV-l induced transcripts in flow cytometry using a ge- nomic probe, digoxigenin-labeled by random prim- ing. and an anti-digoxigenin fluoresceinated anti- serum. Cytospin preparations were made from the cell suspensions used for flow cytometry and the intracellular distribution of the target RNA was eval- uated by fluorescence and confocal microscopy. We have also tested different procedures for double la- beling HIV-RNA together with either surface mark- ers (CD4. CD3) or virus induced proteins (~24) (Costigliola et al., 1992; Gadol et al.. 1994). The procedure was applied to a panel of chroni- cally infected cell lines and to an acutely infected cell line mimicking normal peripheral blood lympho- cytes in susceptibility to HIV- 1. The potential appli- cations of this method to clinical samples are dis- cussed. 2. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJI aterials and methods Different fixation procedures were tested, yielding comparable results with regard to the hybridization signal. In the first series of experiments we used the fixation procedure previously described by Lalli et al. 1992): after two washes with PBS at 4°C the cell samples were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature, then spun at 1000 X g for 5 min and thoroughly resuspended in 70% ethanol. The cells were then stored at -20°C at a concentration of 4 X 106/ml. For a series of samples we also prepared parallel aliquots of cells which only underwent fixation with PFA: a first fixation step with 4% PFA for 10 min was followed by treatment with 1% PFA for 72 h in order to completely abrogate HIV infectivity (Aloisio and Nicholson, 1990; Cory et al., 1990; Nicholson et al., 1993). The cells were then stored at 4°C in diethyl pyrocarbonate (DEPC)-treated PBS and used within I month. 2.1.1. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG IV-I’ cell lines Initially we used both lymphoblastoid or monocy- toid HIV- zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON   cell lines as target cells. The lym- phoblastoid cell line was the MT4SI. a subclone selected as being a high HIV-l producer obtained from the MT-4 cell line (HTLV-1 transformed hu-  man T cells: Larder et al., 1989; Pauwels et al.. 1987) which underwent superinfection with HIV- I. The monocytoid cell line was the Ul cell line. a subclone of U-937 chronically infected with HIV-1 and containing two copies of provirus per cell (Folks et al.. 1987). 2.1.2. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA cute infection of an uninfected cell line For investigations of acute infection we utilized the A3.01 cell line, HAT-sensitive derivative of CEM. CD4+. CD62L+, CD5+, CD71+, CD25-, sensitive to infection with HIV-l and susceptible to cytopathic effects when infected (Folks et al., 1985). The A3.01 cell line, which mimics normal peripheral blood lymphocytes in susceptibility to HIV-l, was infected with NL4-3 free viral particles (TCID,, 6.4). This HIV-l strain was prepared by Adachi and coworkers: following transfection of SW480 and A3.01 cells with molecularly cloned DNA (total genomic DNA from NY5 and LAV: 9709 bp) the virus particles were harvested and passaged into A3.01 cells (Adachi et al., 1986). In our protocol the cells were collected on days 2, 4. 7 and 9 post infection in order to monitor the infection kinetically in terms of the percentage of HIV-RNA positive cells. For each time point. 10’ A3.01 cells in 8 ml of culture medium were exposed to 500 ~1 of supematant from a previous infection which had yielded a ~34 level higher than 1000 pg/ml. After 24 h the virus was removed by wash- ing the cells three times with supplemented Hanks’ V buffer (Hanks’ V buffer supplemented with 14 mM NaHCO, and 11 mM glucose) and this was taken as day 0 of the infection schedule. The infec- tion of these cell lines was also monitored by testing the level of p24 (Coulter HIV-l p24 antigen assay) released in the supematant of the cultures. 2.2. Fluorescent in situ hybridkation The double-stranded DNA probe was obtained from pMN: a molecular clone of HIV- 1, a kind gift of Franc0 Lori. from the Laboratory of Robert Gallo, NIH, Bethesda. pMN contains the whole sequence (9746 nucleotides) of the MN strain of HIV-l, first isolated in 1984 (Gallo et al., 1984) from a pediatric patient with AIDS and grown in a permanent T cell line. The restriction enzyme pattern of MN in com- parison with other HIV-l isolates was described by Wong-Staal et al. (1985). pMN (insert plus vector> is 16 kb long; it is biologically active and is both lymphocytotropic and monocyte/macrophagetropic. To obtain the probe, pMN was digoxigenin la- beled by means of a standard random priming method (Boehringer, Mannheim): after denaturation of the template DNA. Klenow enzyme was used to synthe- size new DNA along the single stranded substrate starting from the 3’-OH end of the annealed primer with incorporation of digoxigenin-dUTP at an aver- age rate of l/22.5 nucleotides. In this way, a hetero- geneous population of probe strands is produced. many with overlapping complementary regions, and this provides signal amplification in hybridization experiments. Probe molecule lengths were in the 100-400 bp range (data not shown). as evaluated by agarose gel electrophoresis, and were of random specificity along the whole sequence of pMN. De- spite HIV-l variability, invariant and highly con- served regions are present in the viral structural genes as well as in the genes for the replicative enzymes and for the regulatory proteins. Further- more. the two causative agents of AIDS, HIV-1 and HIV-2, share a certain degree of sequence homology (Guyader et al., 1987). For this reason the random primed pMN probe is suitable for hybridizing cells infected with various strains of HIV-1 and HIV-2. Basically, the FISH procedure was adapted from a previously published procedure (Lalli et al., 1992) with minor modifications. Moreover, as described in detail below, the procedure itself was slightly modi- fied for the double labeling. Aliquots of 2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTS   IO5 fixed cells per sample were used and initially treated with 10% DEPC in ethanol (1 ~1 in 50 ~1 sample) for 1 h to inhibit endogenous ribonucleases. The cells were then spun down by centrifuging for 2.5 min in a table top microfuge at 3400 X g and resuspended in 50 ~1 of 0.5% Tween 20 in PBS, for 5 min at room temperature. After permeabilization the samples underwent a pre-hy- bridization step by adding 1 vol. of 20 X SSC and 2 ~01s. of deionized formamide to each tube, then spinning down for 2.5 min. The cells were resuspended in 20 ~1 of hybridiza- tion buffer (50% formamide. 5 X SSC. 5 X Den- hardt’s, 0.5% SDS) containing 50 ng of freshly denatured genomic probe and left to hybridize  17 overnight at 48°C. Stringency washings were started tometer equipped with an argon ion laser tuned at by the addition of 100 ~1 of 0. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA  X SSC, then the 488 nm. 200 mW. Green fluorescence (FIT0 was pelleted cells were resuspended in 100 ~1 of 0.1 X collected through a 530/30 nm band-pass (BP) filter SSC and incubated for 45 min at 48°C. The hy- and red fluorescence (PE) through a 585/42 nm BP bridization of the HIV-l probe was to RNA and not filter. Electronic compensation was used between the DNA since the cells were not subjected to denatura- fluorescence channels to remove residual spectra1 tion and DNA strand separation was prevented. overlaps. The detection phase was preceded by a blocking step to prevent non-specific binding. In this cells were saturated by a 15 min incubation at room temperature in 1% blocking buffer (Boehringer, Mannheim) in 0.1 M maleic acid, 0.15 M NaCl pH 7.5. Then a 1 lo00 dilution of a FITC-conjugated F(ab’), sheep anti-digoxigenin antiserum (Boeh- ringer, Mannheim) was added and left for 30 min at room temperature with gentle shaking. This dilution of the antiserum was chosen on the basis of the best signal to noise ratio. Before cytofluorimetric analysis the cells were washed twice in 0.5% Tween 20 in PBS, and finally resuspended in 200 ~1 of PBS. 2.2.1. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ouble labeling Cell aggregates were gated out by forward and right angle light signals and 5000-10000 events were collected for each sample. Fluorescence data were displayed on a four-decade log scale. with 1024 channel resolution. For comparison between experi- ments a fluorescence standard (CaliBrite microbeads, Becton Dickinson) was employed, so that the median fluorescence intensity of both FITC- and PE-labeled beads was set to channel no. 500. In some experi- ments, fluorescein quantitative standard beads (Flow Cytometry Standards. Research Triangle Park. NC) containing 0, 8.7 X IO’. 4.7 X 10’. 1.4 X 10’ and 4.5 X IO5 molecules of equivalent soluble fluo- rochrome (MESF) were used to quantify the hy- bridization signal. constructing a standard curve of fluorescence. Staining of intracellular p24 was performed on the A3.01 infected cells at the end of the hybridization procedure, by adding a mouse anti-p24 monoclonal antibody (Du Pont, Brussels, Belgium) at a concen- tration of 10 mg/ml. at the same time as the anti-DIG FITC antiserum. Detection was achieved by an addi- tional incubation with an antimouse PE conjugated antiserum (Caltag Laboratories, S. Francisco, CA), diluted l/20. Fluorescence histogram statistics were calculated by the instrument software and median fluorescence intensity values, converted into linear fluorescence channels. were used to evaluate the signal to noise ratios of samples relative to controls. 2.4. Cwfocal microscopy We also tested the possibility of staining surface markers in association with FISH. The preservation of good membrane reactivity was ensured by use of the following procedure: (1) labeling of CDs (CD4, CD3) on either fresh or PFA fixed cells using bi- otinylated antibodies (30 min at room temperature): (2) additional fixation with buffered 10% formalin ( 15 min at room temperature): (3) hybridization with a digoxigenin-labeled probe: (4) immunological de- tection with an FITC-conjugated anti-digoxigenin an- tiserum and PE-streptavidin. The localization of the hybridization signal was imaged by a Phoibos IOOO-Sarastro (Molecular Dy- namics, USA) confocal laser scanning microscope mounted on a Nikon Optiophot microscope (Nikon. Tokyo, Japan). Samples were observed with a 60 X 1.4 numerical aperture planapochromat oil immer- sion objective lens. 2.3. Flow cytometric analysis Samples were analyzed with a FACStar PLUS (Becton Dickinson. Mountain View, CA) flow cy- For image acquisition the samples were excited with the green (488 nm> line of a 25 mW multiline argon zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP on laser. The laser power was tuned at 10 mW to obtain the highest light stability and the laser beam was attenuated to 30% of transmission with a neutral density filter to limit bleaching of the FITC fluorescence. The emission signal was observed us- ing a dichroic mirror (500 nm) and a cut-off filter (530 nm). The detector was a photomultiplier and the pinhole in the front of the detector was 50 mm in  R.M. Bar? et al. /Journal of Immurtologicai Methods 193 1996) 167-176 171 Fig. I. Flow cytometric analysis of HIV-l RNA in chronically infected MT4SI cells after fluorescent in situ hybridization in suspension. FITC fluorescence (plotted on the abscissa) is ex- pressed on a logarithmic scale using a 1024 channel instrument. a: no probe hybridization cells. b: cells hybridized with an irrelevant probe (pBR328). c: cells hybridized with the specific probe @MN). d: RNAse treated cells hybridized with pMN. size. Optical sections were obtained at increments of 0.3 mm in the ; axis and stored on the computer (Silicon Graphics, Mountain View, CA; Indigo XS- 24 workstation), with a scanning mode format of 512 X 512 pixels. Image processing (Gaussian filtering, vectoriza- tion and volume rendering) was performed on sec- tion series using Image Space software (Molecular Dynamics) to obtain spatial projection. The distribu- tion of the hybridization signal was investigated recording both fluorescence and phase-contrast sig- nal, elaborated independently to obtain the transmit- ted and confocal patterns, superimposed and then simultaneously analysed. 3. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON esults 3.1. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF ell fixation Samples fixed with 4% PFA and stored in 70% ethanol proved to be suitable for hybridization for several months, since intracellular RNA was pre- served. Nevertheless, in some experiments cellular integrity was compromised by the use of ethanol so that only a few cells could be recovered to perform cytofluorimetric analysis. The fixation procedure with PFA alone was found to better preserve cell morphology (as detected both by microscopy and by the light scatter signals in flow cytometry) and was suitable for double labeling of surface CDs. Fig. 2. Detection of HIV-l RNA in the MT4SI cell line by fluorescent in situ hybridization. The cytoplasmatic distribution of pMN hybridized RNA was visualized with confocal microscopy by superimposing phase contrast and fluorescence signals (white spots).
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