Vol. 65, No. 4
JOURNAL OF VIROLOGY, Apr. 1991, p. 1812-1822 0022-538X/91/041812-11$02.00/0 Copyright C 1991, American Society for Microbiology
In Vitro Infection of Natural Killer Cells with Different Human Immunodeficiency Virus Type 1 Isolates JIHED CHEHIMI,1* SANTU BANDYOPADHYAY,l KESH PRAKASH,' BICE PERUSSIA,2 NASSEF F. HASSAN,' HISASHI KAWASHIMA,1 DONALD CAMPBELL,' JACKI KORNBLUTH,3 AND STUART E. STARR' The Division of Infectious Diseases and Immunology, The Joseph Stokes, Jr. Research Institute of the Children's Hospital of Philadelphia,' and The Wistar Institute of Anatomy and Biology,2 Philadelphia, Pennsylvania 19104, and Department of Immunology, The University of Arkansas, Little Rock, Arkansas 722053 Received 11 October 1990/Accepted 28 December 1990
Natural killer (NK) cells are a discrete subset of leukocytes, distinct from T and B lymphocytes. NK cells mediate spontaneous non-MHC-restricted killing of a wide variety of target cells without prior sensitization and appear to be involved in initial protection against certain viral infections. Depressed NK cell-mediated cytotoxicity, one of the many immunological defects observed in AIDS patients, may contribute to secondary virus infections. Here we report that clonal and purified polyclonal populations of NK cells, which expressed neither surface CD4 nor CD4 mRNA, were susceptible to infection with various isolates of human immunodeficiency virus type 1 (HIV-1). Viral replication was demonstrated by detection of p24 antigen intracellularly and in culture supernatants, by the presence of HIV DNA within infected cells, and by the ability of supernatants derived from HIV-infected NK cells to infect peripheral blood mononuclear cells or CD4+ cell lines. Infection of NK cells was not blocked by anti-CD4 or anti-FcyRIII monoclonal antibodies. NK cells from HIV-infected and uninfected cultures were similar in their ability to lyse three different target cells. Considerable numbers of cells died in HIV-infected NK cell cultures. These results suggest that loss of NK cells in AIDS patients is a direct effect of HIV infection but that reduced NK cell function involves another mechanism. The possibility that NK cells serve as a potential reservoir for HIV-1 must be considered.
lation between low levels of NK cell activity and the severity of viral infections in immunocompromised hosts. For example, in immunosuppressed recipients of a bone marrow transplant, a correlation exists between fatal cytomegalovirus (CMV) infection and failure to develop NK cell and cytotoxic T-lymphocyte-mediated cytotoxicity (40). NK cell-mediated antibody-dependent cellular cytotoxicity may play an important role in the pathogenesis of HIV-1 infection (49); however, conflicting data have appeared concerning NK cell activity in AIDS patients (reviewed in reference 5), although in most studies decreased NK cell-mediated cytotoxicity was observed. Reported differences among studies may be attributed to different patient populations, different target cells, or different assay conditions. Nevertheless, impaired NK cell activity is one of the many immunological defects observed in patients with AIDS and related syndromes, and its cause remains unclear. We show for the first time in this report that purified NK cells, which neither bear the CD4 antigen on their surface nor contain CD4 mRNA, can be productively infected in vitro by various HIV-1 isolates.
Human immunodeficiency virus (HIV), the causative agent of AIDS, can devastate the immune system. Although the pathogenesis of AIDS is clearly related to the tropism of
the virus for CD4+ T lymphocytes (13, 22, 26, 37, 38, 46), a wide variety of cell types, all bearing CD4, can be infected by HIV type 1 (HIV-1) (reviewed in reference 43). However, there is mounting evidence that CD4- cells are also susceptible to HIV-1 infection in vivo and in vitro (7), raising the possibility that HIV can enter cells via a receptor other than the CD4 glycoprotein. Recent studies have shown that HIV-1 infection of some CD4+ cells can occur via a CD4independent mechanism (11, 24, 51). Taken together, these studies extended the original host range of HIV-1 and raised the possibility that multiple receptors for HIV-1 exist. The nature and role of humoral and cellular immune mechanisms against HIV have not been fully characterized. Natural killer (NK) cells represent a discrete leukocyte subset distinct from T and B lymphocytes (reviewed in reference 48). NK cells have been identified in peripheral blood as a population of T-cell-receptor-negative cells that express CD16 (Fc-yRIII, a low-affinity receptor for the Fc fragment of immunoglobulin G [IgG]) and CD56 (NKH1) cell surface markers. NK cells mediate two forms of cytotoxicity: (i) lysis of tumor cells and virus-infected cells in a non-MHC-restricted manner and without prior sensitization; (ii) through CD16, NK cells can lyse IgG antibody-coated target cells; this mechanism is known as antibody-dependent cellular cytotoxicity. NK cells may provide the first line of defense against certain viruses before activation of specific humoral and cellular immune mechanisms. Decreased NK cell activity has been reported in patients with a variety of different diseases (48). Several studies have shown a corre*
MATERIALS AND METHODS NK cells. All experiments were performed on clonal and polyclonal populations of human peripheral blood NK cells. Polyclonal peripheral blood NK cells were prepared by a method similar to that previously described by Perussia et al. (32). Briefly, peripheral blood mononuclear cells (PBMC) from healthy adult donors who were seronegative for hepatitis B virus and HIV were isolated by centrifugation over Ficoll-Hypaque. Monocytes were partially depleted by two cycles of adherence on plastic. Peripheral blood lymphocytes (2.5 x 105/ml in RPMI 1640 [Flow Laboratories, Inc., McLean, Va.]) containing 10% fetal bovine serum [FBS]
Corresponding author. 1812
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[Hyclone Laboratories, Logan, Utah]) were then cocultured with 50-Gy-irradiated, RPMI 8866 B-lymphoblastoid cells at a ratio of 5:1 for 10 days at 37°C. Viable cells were collected, and NK cells were enriched by density gradient centrifugation after sensitization of the lymphocytes with a mixture of anti-T monoclonal antibodies (MAbs) B36.1 (anti-CD5, IgG2b) (35) and OKT3 (anti-CD3 purchased from the American Type Culture Collection, Rockville, Md.) and antimonocyte B52.1 (anti-CD14, IgM) (14), followed by indirect rosetting with CrCl3-treated goat anti-mouse Ig-coated sheep erythrocytes. Enriched NK cell preparations were further depleted of minimal numbers of contaminating T or B cells and monocytes by two cycles of antibody-plus-complementmediated lysis as described previously (9). The following antibodies were used: OKT4 (anti-CD4 from the American Type Culture Collection) for depletion of CD4+ cells, Bi (anti-CD20; Coulter Immunology, Hialeah, Fla.), and B52.1 for depletion of monocytes. Viable cells were collected by centrifugation over Ficoll-Hypaque. The generation of NK clone 3.3 has been previously reported by Kornbluth et al. (23). Virus. Culture supernatants of the following HIV-1 isolates were used: IIIB, grown in HUT78 cells (39); SF2, formerly called ARV-2 (10), grown in Sup-Ti cells; SF162 (21), grown in phytohemagglutin-interleukin-2 (PHA-IL-2)stimulated PBMC (kindly provided by J. A. Levy, University of California San Francisco); and WMJ1 (20), grown in Sup-Ti cells (kindly provided by J. A. Hoxie, University of Pennsylvania); as well as five fresh isolates from pediatric AIDS patients seen at the Children's Hospital of Philadelphia (Ml, M2, M3, 43NA, and 44NA). Ml, M2, and M3 were isolated from monocytes, and 43NA and 44NA were isolated from T cells. The detailed cellular tropisms of these isolates are still under investigation. All fresh isolates were used at passage 1. Supernatants were cleared of cells by centrifugation, filtered through 0.45-,um-pore-size filters, and stored in liquid nitrogen until use. Cells and supernatants were tested routinely for mycoplasma contamination and found to be negative. Virus stocks were titered for p24 core antigen production by an antigen capture assay (Coulter Immunology) and by tissue culture infective dose titrations with Sup-Ti cells, HUT78 cells, or PHA-IL-2-stimulated PBMC. Surface marker analysis. NK cell populations were analyzed for surface markers by flow cytometry as previously described (9). Fluorescein isothiocyanate conjugates of the following MAbs were used: Bi; B33.1 (anti-HLA-DR nonpolymorphic determinant; kindly provided by G. Trinchieri, The Wistar Institute) (35); OKT3 and B36.1 for detection of T cells; B52.1; B73.1 (anti-CD16 antigen) (31, 33); 3G8, which recognizes a different epitope of the CD16 molecule (17); and B159.1 (anti-CD56, provided by G. Trinchieri). The CD4 antigen was detected by indirect immunofluorescence with the Leu-3a (Becton Dickinson, Mountain View, Calif.) and OKT4 anti-CD4 MAbs. Irrelevant isotype-matched MAbs (Becton Dickinson) were used as controls. To exclude the possibility of T-cell outgrowth in the polyclonal peripheral blood NK cells, we analyzed IL-2-cultured cells for the presence of T cells after 10 days in culture. Cells were analyzed with a Cytofluorograph System 50 H connected to a 2150 data handling system (Ortho Diagnostic Systems Inc., Westwood, Mass.). Forward and right-angle light scatter was used to establish appropriate gates on the cells, excluding nonviable cells. The fluorescence distribution of 5,000 to 10,000 cells was accumulated for analysis. Cells were considered positive when their fluorescence intensity exceeded
HIV-1 INFECTION OF NK CELLS
1813
the threshold at which 99% of cells stained with control MAb had lower fluorescence intensity. Detection of CD4 mRNA by Northern (RNA) blot analysis. Total RNA from the clonal and polyclonal populations of human NK cells was extracted by the method previously described by Favaloro et al. (16). RNA (10 ,ug) was separated on a 1% agarose-0.75 M formaldehyde gel and transferred to nitrocellulose (Optibind; Schleicher and Schuell, Inc., Keene, N.H.). Prehybridization was done at 42°C for 1 h in 50% formamide (vol/vol)-S5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-5x Denhardt solution0.1% sodium dodecyl sulfate (SDS)-100 ,ug of sonicated denatured salmon testes DNA per ml. Hybridization was done at 42°C for 24 h with 1.5 x 106 cpm of 32P-labeled CD4 probe per ml. The probe used for the detection of the CD4 mRNA was the full-length 3.0-kb cDNA, pT4B (kindly provided by A. Srinivasan, The Wistar Institute) (27). Washing was done at room temperature for 15 min (three times, 5 min each) with 2 x SSC-0. 1% SDS and then twice at 42°C (30 min each) with 2x SSC-0.1% SDS. The membrane was dried and exposed for autoradiography at -70°C to an XRP-5 (Kodak) X-ray film and an intensifying screen. ,B-Actin probe was used to verify the integrity of the RNA samples. Cell culture and virus infection. Polyclonal populations of purified NK cells from peripheral blood were incubated at 37°C in RPMI 1640-10% FBS plus 100 U of IL-2 (Pharmacia Inc., Piscataway, N.J.) per ml. NK clone 3.3 was maintained in RPMI 1640 containing 15% FBS plus 15% IL-2-conditioned medium (Lymphocult T-LF; Biotest, Denville, N.J.) with biweekly feeding and splitting back to a density of 3 x 105 cells per ml. Polyclonal and monoclonal NK cells (2 x 106 to 3 x 106) in 15-ml conical plastic centrifuge tubes were inoculated with different cell-free HIV isolates at a multiplicity of infection of 0.01 50% tissue culture infective dose. After 3 to 4 h of incubation at 37°C, cells were washed five to six times with serum-free RPMI. The final wash was done in 1.5 ml of medium and saved for p24 antigen determinations. Cells were resuspended in culture medium and incubated for an additional 18 h. The cells were then washed twice and maintained in 25-cm2 tissue culture flasks or in 12-well tissue culture plates. Detection of HIV infection. At different time intervals, culture supernatants and cells were harvested for p24 core antigen determinations by an antigen capture assay (Coulter Immunology). Cells were washed three times in phosphatebuffered saline (PBS) and lysed. Assays were done according to the manufacturer's instructions. The A450 cutoff value for each p24 assay was calculated by using the mean of results obtained with cells and supernatants of uninfected control cultures adjusted according to the manufacturer. Cutoff values ranged from 0.120 to 0.160. The percentage of infected cells was determined by indirect immunofluorescence, using MAb to p24 core protein (Du Pont Co., Wilmington, Del.) and heat-inactivated serum from an adult patient with AIDS. Cells were washed and fixed in suspension with 95% methanol for 90 min at -70°C. Cells were then washed with PBS containing 10% goat serum and incubated with a 1:10 dilution of MAb to p24 or with a 1:50 dilution of the HIV-seropositive serum for 1 h at 37°C. Cells were washed twice in a large volume of PBS-10% goat serum and 0.05% Tween and incubated for 1 h with a 1:80 dilution of fluorescein isothiocyanate-goat F(ab')2 anti-mouse Ig (Organon Teknika Corp., Durham, N.C.) or with a 1:50 dilution of fluorescein isothiocyanate-goat F(ab')2 anti-human IgG (Organon Teknika) for 45 min at 37°C. After extensive washing,
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cells were analyzed by flow cytometry and examined by fluorescence microscopy. Cell viability. Cell viability was determined by the trypan blue dye exclusion method. HIV-infected and uninfected polyclonal and clonal populations of NK cells were counted every 3 to 4 days, and the percent cell viability was determined. Detection of HIV DNA in infected NK cells. DNA from uninfected and infected NK cells was extracted by the method previously described by Blin and Stafford (3), digested with the restriction endonuclease Sacl, and electrophoresed in a 1% agarose gel. The digests were transferred to a nitrocellulose membrane and hybridized to a nicktranslated 32P-labeled full-length 9.0-kb HIV probe derived from the clone HXB2 (41), kindly provided by P. Reddy, The Wistar Institute. Prehybridization was done at 55°C for 1 h as described above for detection of CD4 mRNA. Hybridization was then done overnight at 55°C as described above with 106 cpm of the 32P-labeled probe per ml. The membrane was washed at 55°C three times for a total of 90 min in 2 x SSC-0.1% SDS and exposed for autoradiography at -70°C to an XRP-5 (Kodak) X-ray film and an intensifying screen. Blocking experiments. Polyclonal peripheral blood NK cells and clone 3.3 cells at a concentration of 5 x 105 cells per ml were incubated for 1 h at 37°C with 25 p.g of MAb 3G8 (anti-FcyRIII) or 10 ,ug of MAb Leu-3a detecting the CD4 molecule and which has been shown to block HIV replication in CD4+ cells (46). The cells were then inoculated with HIV-1 isolate IIIB or SF162 and incubated for an additional 2 h. The cells were washed (four times) with serum-free medium, the appropriate MAb was added at the same concentration as previously, and the cells were incubated at 37°C. At days 4 and 10, cells and supernatants were harvested for p24 antigen determinations. Syncytium assays. Filtered cell-free supernatants from HIV-IIIB-infected NK clone 3.3 cells, containing approximately 0.8 to 1.5 ng of p24 antigen, were added to the indicator cells. Four different indicator cells were used: PHA-IL-2-stimulated PBMC from healthy donors; two CD4+ T cell lines, HUT78 and MT-4; and the monoblastoid cell line U937. In parallel, the same indicator cells were infected with supernatant from HUT78 cells chronically infected with IIIB. HIV production was monitored at various time postinfection by the appearance of p24 antigen in the supernatants and in the cells. Cells were checked daily for cytopathic effects (CPE) and syncytia formation. NK cell-mediated cytotoxicity assays. Cytotoxicity assays with K562 cells, CMV-infected fibroblasts (CMV-FS4), and HUT78 cells chronically infected with IIIB (IIIB-HUT78) were performed as described previously (9). Briefly, uninfected and HIV-1-infected polyclonal peripheral blood and clone 3.3 NK cells were used as effector cells. NK cells infected with different HIV strains (IIIB, SF2, and WMJ1) as described above were harvested 8 to 10 days after HIV infection, washed twice, and kept for 2 h in IL-2-free RPMI medium before use. Cells were then washed one more time and used in cytotoxicity assays. CMV-FS4 were prepared as previously described (1). Briefly, human embryonic foreskin fibroblasts (FS4 strain, kindly provided by J. Vilcek, New York Medical Center, New York, N.Y.) were maintained in Eagle modified minimal essential medium (Flow Laboratories) supplemented with 10% heat-inactivated FBS, 2% vitamins, and 2 mM glutamine. FS4 cells were used at passage 16 to 24. Monolayers of FS4 cells in 75-cm2 flasks were infected with human CMV strain AD-169 at a multiplicity of infection of -0.1. When 90% of cells exhibited
TABLE 1. Phenotypic characterization of NK cellsa MAbs used
% Positive cells NK clone 3.3
Polyclonal NK cellsb
CD3 CD4 CD5 CD14 CD16 CD20 CD56
0 0 0 0 >98 0 >92
95
HLA-DR
>98
>96
a Results are expressed as percentage of positive cells as determined by cytofluorographic analysis. Isotype-matched control MAb stained less than 1% of cells. Values shown are representative of four to eight separate experiments. Standard deviations were within 2% of mean values (not shown). b Polyclonal populations of peripheral blood NK cells were prepared as described in Materials and Methods. Polyclonal peripheral blood NK cells were analyzed by flow cytofluorometry for the presence of T cells after 10 days of culture in IL-2; none were detected (0o CD3, 0% CD4,
JOURNAL OF VIROLOGY, Apr. 1991, p. 1812-1822 0022-538X/91/041812-11$02.00/0 Copyright C 1991, American Society for Microbiology
In Vitro Infection of Natural Killer Cells with Different Human Immunodeficiency Virus Type 1 Isolates JIHED CHEHIMI,1* SANTU BANDYOPADHYAY,l KESH PRAKASH,' BICE PERUSSIA,2 NASSEF F. HASSAN,' HISASHI KAWASHIMA,1 DONALD CAMPBELL,' JACKI KORNBLUTH,3 AND STUART E. STARR' The Division of Infectious Diseases and Immunology, The Joseph Stokes, Jr. Research Institute of the Children's Hospital of Philadelphia,' and The Wistar Institute of Anatomy and Biology,2 Philadelphia, Pennsylvania 19104, and Department of Immunology, The University of Arkansas, Little Rock, Arkansas 722053 Received 11 October 1990/Accepted 28 December 1990
Natural killer (NK) cells are a discrete subset of leukocytes, distinct from T and B lymphocytes. NK cells mediate spontaneous non-MHC-restricted killing of a wide variety of target cells without prior sensitization and appear to be involved in initial protection against certain viral infections. Depressed NK cell-mediated cytotoxicity, one of the many immunological defects observed in AIDS patients, may contribute to secondary virus infections. Here we report that clonal and purified polyclonal populations of NK cells, which expressed neither surface CD4 nor CD4 mRNA, were susceptible to infection with various isolates of human immunodeficiency virus type 1 (HIV-1). Viral replication was demonstrated by detection of p24 antigen intracellularly and in culture supernatants, by the presence of HIV DNA within infected cells, and by the ability of supernatants derived from HIV-infected NK cells to infect peripheral blood mononuclear cells or CD4+ cell lines. Infection of NK cells was not blocked by anti-CD4 or anti-FcyRIII monoclonal antibodies. NK cells from HIV-infected and uninfected cultures were similar in their ability to lyse three different target cells. Considerable numbers of cells died in HIV-infected NK cell cultures. These results suggest that loss of NK cells in AIDS patients is a direct effect of HIV infection but that reduced NK cell function involves another mechanism. The possibility that NK cells serve as a potential reservoir for HIV-1 must be considered.
lation between low levels of NK cell activity and the severity of viral infections in immunocompromised hosts. For example, in immunosuppressed recipients of a bone marrow transplant, a correlation exists between fatal cytomegalovirus (CMV) infection and failure to develop NK cell and cytotoxic T-lymphocyte-mediated cytotoxicity (40). NK cell-mediated antibody-dependent cellular cytotoxicity may play an important role in the pathogenesis of HIV-1 infection (49); however, conflicting data have appeared concerning NK cell activity in AIDS patients (reviewed in reference 5), although in most studies decreased NK cell-mediated cytotoxicity was observed. Reported differences among studies may be attributed to different patient populations, different target cells, or different assay conditions. Nevertheless, impaired NK cell activity is one of the many immunological defects observed in patients with AIDS and related syndromes, and its cause remains unclear. We show for the first time in this report that purified NK cells, which neither bear the CD4 antigen on their surface nor contain CD4 mRNA, can be productively infected in vitro by various HIV-1 isolates.
Human immunodeficiency virus (HIV), the causative agent of AIDS, can devastate the immune system. Although the pathogenesis of AIDS is clearly related to the tropism of
the virus for CD4+ T lymphocytes (13, 22, 26, 37, 38, 46), a wide variety of cell types, all bearing CD4, can be infected by HIV type 1 (HIV-1) (reviewed in reference 43). However, there is mounting evidence that CD4- cells are also susceptible to HIV-1 infection in vivo and in vitro (7), raising the possibility that HIV can enter cells via a receptor other than the CD4 glycoprotein. Recent studies have shown that HIV-1 infection of some CD4+ cells can occur via a CD4independent mechanism (11, 24, 51). Taken together, these studies extended the original host range of HIV-1 and raised the possibility that multiple receptors for HIV-1 exist. The nature and role of humoral and cellular immune mechanisms against HIV have not been fully characterized. Natural killer (NK) cells represent a discrete leukocyte subset distinct from T and B lymphocytes (reviewed in reference 48). NK cells have been identified in peripheral blood as a population of T-cell-receptor-negative cells that express CD16 (Fc-yRIII, a low-affinity receptor for the Fc fragment of immunoglobulin G [IgG]) and CD56 (NKH1) cell surface markers. NK cells mediate two forms of cytotoxicity: (i) lysis of tumor cells and virus-infected cells in a non-MHC-restricted manner and without prior sensitization; (ii) through CD16, NK cells can lyse IgG antibody-coated target cells; this mechanism is known as antibody-dependent cellular cytotoxicity. NK cells may provide the first line of defense against certain viruses before activation of specific humoral and cellular immune mechanisms. Decreased NK cell activity has been reported in patients with a variety of different diseases (48). Several studies have shown a corre*
MATERIALS AND METHODS NK cells. All experiments were performed on clonal and polyclonal populations of human peripheral blood NK cells. Polyclonal peripheral blood NK cells were prepared by a method similar to that previously described by Perussia et al. (32). Briefly, peripheral blood mononuclear cells (PBMC) from healthy adult donors who were seronegative for hepatitis B virus and HIV were isolated by centrifugation over Ficoll-Hypaque. Monocytes were partially depleted by two cycles of adherence on plastic. Peripheral blood lymphocytes (2.5 x 105/ml in RPMI 1640 [Flow Laboratories, Inc., McLean, Va.]) containing 10% fetal bovine serum [FBS]
Corresponding author. 1812
VOL. 65, 1991
[Hyclone Laboratories, Logan, Utah]) were then cocultured with 50-Gy-irradiated, RPMI 8866 B-lymphoblastoid cells at a ratio of 5:1 for 10 days at 37°C. Viable cells were collected, and NK cells were enriched by density gradient centrifugation after sensitization of the lymphocytes with a mixture of anti-T monoclonal antibodies (MAbs) B36.1 (anti-CD5, IgG2b) (35) and OKT3 (anti-CD3 purchased from the American Type Culture Collection, Rockville, Md.) and antimonocyte B52.1 (anti-CD14, IgM) (14), followed by indirect rosetting with CrCl3-treated goat anti-mouse Ig-coated sheep erythrocytes. Enriched NK cell preparations were further depleted of minimal numbers of contaminating T or B cells and monocytes by two cycles of antibody-plus-complementmediated lysis as described previously (9). The following antibodies were used: OKT4 (anti-CD4 from the American Type Culture Collection) for depletion of CD4+ cells, Bi (anti-CD20; Coulter Immunology, Hialeah, Fla.), and B52.1 for depletion of monocytes. Viable cells were collected by centrifugation over Ficoll-Hypaque. The generation of NK clone 3.3 has been previously reported by Kornbluth et al. (23). Virus. Culture supernatants of the following HIV-1 isolates were used: IIIB, grown in HUT78 cells (39); SF2, formerly called ARV-2 (10), grown in Sup-Ti cells; SF162 (21), grown in phytohemagglutin-interleukin-2 (PHA-IL-2)stimulated PBMC (kindly provided by J. A. Levy, University of California San Francisco); and WMJ1 (20), grown in Sup-Ti cells (kindly provided by J. A. Hoxie, University of Pennsylvania); as well as five fresh isolates from pediatric AIDS patients seen at the Children's Hospital of Philadelphia (Ml, M2, M3, 43NA, and 44NA). Ml, M2, and M3 were isolated from monocytes, and 43NA and 44NA were isolated from T cells. The detailed cellular tropisms of these isolates are still under investigation. All fresh isolates were used at passage 1. Supernatants were cleared of cells by centrifugation, filtered through 0.45-,um-pore-size filters, and stored in liquid nitrogen until use. Cells and supernatants were tested routinely for mycoplasma contamination and found to be negative. Virus stocks were titered for p24 core antigen production by an antigen capture assay (Coulter Immunology) and by tissue culture infective dose titrations with Sup-Ti cells, HUT78 cells, or PHA-IL-2-stimulated PBMC. Surface marker analysis. NK cell populations were analyzed for surface markers by flow cytometry as previously described (9). Fluorescein isothiocyanate conjugates of the following MAbs were used: Bi; B33.1 (anti-HLA-DR nonpolymorphic determinant; kindly provided by G. Trinchieri, The Wistar Institute) (35); OKT3 and B36.1 for detection of T cells; B52.1; B73.1 (anti-CD16 antigen) (31, 33); 3G8, which recognizes a different epitope of the CD16 molecule (17); and B159.1 (anti-CD56, provided by G. Trinchieri). The CD4 antigen was detected by indirect immunofluorescence with the Leu-3a (Becton Dickinson, Mountain View, Calif.) and OKT4 anti-CD4 MAbs. Irrelevant isotype-matched MAbs (Becton Dickinson) were used as controls. To exclude the possibility of T-cell outgrowth in the polyclonal peripheral blood NK cells, we analyzed IL-2-cultured cells for the presence of T cells after 10 days in culture. Cells were analyzed with a Cytofluorograph System 50 H connected to a 2150 data handling system (Ortho Diagnostic Systems Inc., Westwood, Mass.). Forward and right-angle light scatter was used to establish appropriate gates on the cells, excluding nonviable cells. The fluorescence distribution of 5,000 to 10,000 cells was accumulated for analysis. Cells were considered positive when their fluorescence intensity exceeded
HIV-1 INFECTION OF NK CELLS
1813
the threshold at which 99% of cells stained with control MAb had lower fluorescence intensity. Detection of CD4 mRNA by Northern (RNA) blot analysis. Total RNA from the clonal and polyclonal populations of human NK cells was extracted by the method previously described by Favaloro et al. (16). RNA (10 ,ug) was separated on a 1% agarose-0.75 M formaldehyde gel and transferred to nitrocellulose (Optibind; Schleicher and Schuell, Inc., Keene, N.H.). Prehybridization was done at 42°C for 1 h in 50% formamide (vol/vol)-S5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-5x Denhardt solution0.1% sodium dodecyl sulfate (SDS)-100 ,ug of sonicated denatured salmon testes DNA per ml. Hybridization was done at 42°C for 24 h with 1.5 x 106 cpm of 32P-labeled CD4 probe per ml. The probe used for the detection of the CD4 mRNA was the full-length 3.0-kb cDNA, pT4B (kindly provided by A. Srinivasan, The Wistar Institute) (27). Washing was done at room temperature for 15 min (three times, 5 min each) with 2 x SSC-0. 1% SDS and then twice at 42°C (30 min each) with 2x SSC-0.1% SDS. The membrane was dried and exposed for autoradiography at -70°C to an XRP-5 (Kodak) X-ray film and an intensifying screen. ,B-Actin probe was used to verify the integrity of the RNA samples. Cell culture and virus infection. Polyclonal populations of purified NK cells from peripheral blood were incubated at 37°C in RPMI 1640-10% FBS plus 100 U of IL-2 (Pharmacia Inc., Piscataway, N.J.) per ml. NK clone 3.3 was maintained in RPMI 1640 containing 15% FBS plus 15% IL-2-conditioned medium (Lymphocult T-LF; Biotest, Denville, N.J.) with biweekly feeding and splitting back to a density of 3 x 105 cells per ml. Polyclonal and monoclonal NK cells (2 x 106 to 3 x 106) in 15-ml conical plastic centrifuge tubes were inoculated with different cell-free HIV isolates at a multiplicity of infection of 0.01 50% tissue culture infective dose. After 3 to 4 h of incubation at 37°C, cells were washed five to six times with serum-free RPMI. The final wash was done in 1.5 ml of medium and saved for p24 antigen determinations. Cells were resuspended in culture medium and incubated for an additional 18 h. The cells were then washed twice and maintained in 25-cm2 tissue culture flasks or in 12-well tissue culture plates. Detection of HIV infection. At different time intervals, culture supernatants and cells were harvested for p24 core antigen determinations by an antigen capture assay (Coulter Immunology). Cells were washed three times in phosphatebuffered saline (PBS) and lysed. Assays were done according to the manufacturer's instructions. The A450 cutoff value for each p24 assay was calculated by using the mean of results obtained with cells and supernatants of uninfected control cultures adjusted according to the manufacturer. Cutoff values ranged from 0.120 to 0.160. The percentage of infected cells was determined by indirect immunofluorescence, using MAb to p24 core protein (Du Pont Co., Wilmington, Del.) and heat-inactivated serum from an adult patient with AIDS. Cells were washed and fixed in suspension with 95% methanol for 90 min at -70°C. Cells were then washed with PBS containing 10% goat serum and incubated with a 1:10 dilution of MAb to p24 or with a 1:50 dilution of the HIV-seropositive serum for 1 h at 37°C. Cells were washed twice in a large volume of PBS-10% goat serum and 0.05% Tween and incubated for 1 h with a 1:80 dilution of fluorescein isothiocyanate-goat F(ab')2 anti-mouse Ig (Organon Teknika Corp., Durham, N.C.) or with a 1:50 dilution of fluorescein isothiocyanate-goat F(ab')2 anti-human IgG (Organon Teknika) for 45 min at 37°C. After extensive washing,
1814
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cells were analyzed by flow cytometry and examined by fluorescence microscopy. Cell viability. Cell viability was determined by the trypan blue dye exclusion method. HIV-infected and uninfected polyclonal and clonal populations of NK cells were counted every 3 to 4 days, and the percent cell viability was determined. Detection of HIV DNA in infected NK cells. DNA from uninfected and infected NK cells was extracted by the method previously described by Blin and Stafford (3), digested with the restriction endonuclease Sacl, and electrophoresed in a 1% agarose gel. The digests were transferred to a nitrocellulose membrane and hybridized to a nicktranslated 32P-labeled full-length 9.0-kb HIV probe derived from the clone HXB2 (41), kindly provided by P. Reddy, The Wistar Institute. Prehybridization was done at 55°C for 1 h as described above for detection of CD4 mRNA. Hybridization was then done overnight at 55°C as described above with 106 cpm of the 32P-labeled probe per ml. The membrane was washed at 55°C three times for a total of 90 min in 2 x SSC-0.1% SDS and exposed for autoradiography at -70°C to an XRP-5 (Kodak) X-ray film and an intensifying screen. Blocking experiments. Polyclonal peripheral blood NK cells and clone 3.3 cells at a concentration of 5 x 105 cells per ml were incubated for 1 h at 37°C with 25 p.g of MAb 3G8 (anti-FcyRIII) or 10 ,ug of MAb Leu-3a detecting the CD4 molecule and which has been shown to block HIV replication in CD4+ cells (46). The cells were then inoculated with HIV-1 isolate IIIB or SF162 and incubated for an additional 2 h. The cells were washed (four times) with serum-free medium, the appropriate MAb was added at the same concentration as previously, and the cells were incubated at 37°C. At days 4 and 10, cells and supernatants were harvested for p24 antigen determinations. Syncytium assays. Filtered cell-free supernatants from HIV-IIIB-infected NK clone 3.3 cells, containing approximately 0.8 to 1.5 ng of p24 antigen, were added to the indicator cells. Four different indicator cells were used: PHA-IL-2-stimulated PBMC from healthy donors; two CD4+ T cell lines, HUT78 and MT-4; and the monoblastoid cell line U937. In parallel, the same indicator cells were infected with supernatant from HUT78 cells chronically infected with IIIB. HIV production was monitored at various time postinfection by the appearance of p24 antigen in the supernatants and in the cells. Cells were checked daily for cytopathic effects (CPE) and syncytia formation. NK cell-mediated cytotoxicity assays. Cytotoxicity assays with K562 cells, CMV-infected fibroblasts (CMV-FS4), and HUT78 cells chronically infected with IIIB (IIIB-HUT78) were performed as described previously (9). Briefly, uninfected and HIV-1-infected polyclonal peripheral blood and clone 3.3 NK cells were used as effector cells. NK cells infected with different HIV strains (IIIB, SF2, and WMJ1) as described above were harvested 8 to 10 days after HIV infection, washed twice, and kept for 2 h in IL-2-free RPMI medium before use. Cells were then washed one more time and used in cytotoxicity assays. CMV-FS4 were prepared as previously described (1). Briefly, human embryonic foreskin fibroblasts (FS4 strain, kindly provided by J. Vilcek, New York Medical Center, New York, N.Y.) were maintained in Eagle modified minimal essential medium (Flow Laboratories) supplemented with 10% heat-inactivated FBS, 2% vitamins, and 2 mM glutamine. FS4 cells were used at passage 16 to 24. Monolayers of FS4 cells in 75-cm2 flasks were infected with human CMV strain AD-169 at a multiplicity of infection of -0.1. When 90% of cells exhibited
TABLE 1. Phenotypic characterization of NK cellsa MAbs used
% Positive cells NK clone 3.3
Polyclonal NK cellsb
CD3 CD4 CD5 CD14 CD16 CD20 CD56
0 0 0 0 >98 0 >92
95
HLA-DR
>98
>96
a Results are expressed as percentage of positive cells as determined by cytofluorographic analysis. Isotype-matched control MAb stained less than 1% of cells. Values shown are representative of four to eight separate experiments. Standard deviations were within 2% of mean values (not shown). b Polyclonal populations of peripheral blood NK cells were prepared as described in Materials and Methods. Polyclonal peripheral blood NK cells were analyzed by flow cytofluorometry for the presence of T cells after 10 days of culture in IL-2; none were detected (0o CD3, 0% CD4,
