INTRODUCTION

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Cytotoxic T-lymphocyte (CTL) responses are a major component of protective immunity against viral infection. During the course of studies on cellular immune responses in human immunodeficiency virus (HIV)-infected people and simian immunodeficiency virus (SIV)-infected rhesus monkeys, our laboratory and others discovered that naive effector controls (from uninfected individuals) could lyse cells expressing HIV or SIV envelope proteins in a major histocompatibility complex (MHC)-unrestricted manner (8, 16, 28, 31, 32). This MHC-unrestricted (UR) lytic activity is mediated primarily by CD4⁺ cells (references 8 and 16 and this study). Thus, CD4⁺ cells not only are the primary targets of infection and virus-mediated apoptosis (26) but also can act as effectors that kill envelope-expressing cells.

Cytolysis mediated by CD4⁺ cells, both MHC restricted and MHC unrestricted, has been observed in many systems. The ₂-microglobulin-deficient mouse is defective for CD8⁺ cell function and shows CD4⁺ cell-mediated lysis that is Fas/FasL mediated and MHC restricted (15, 35). CD4⁺-mediated lysis is of primary importance in controlling murine herpes simplex infection (13). From people vaccinated with HIV gp120, HIV-specific CD4⁺ clones that lyse target cells in both MHC-restricted and -unrestricted manners were isolated (8, 16, 23, 28, 36).

There is now compelling evidence that HIV envelope-CD4 interactions are central to pathogenic mechanisms in HIV-1 infection, including initiating virus attachment to CD4⁺ cells, syncytium formation, and syncytium-independent cytopathic effects (23). The UR lysis that we observe is mediated by envelope-CD4 cell interactions. However, it is only partially blocked by soluble CD4 or antibody to CD4, indicating that other cell-cell interactions are required for lysis. Cell death as a consequence of Fas or tumor necrosis factor receptor signaling (4) is similar to UR lysis in that both are EGTA insensitive. It is possible that UR lysis also occurs through Fas or tumor necrosis factor receptor signaling pathways.

In an effort to define the role of UR lysis in the SIV/rhesus model for AIDS, we screened 56 uninfected rhesus macaques and established the prevalence and magnitude of UR lytic activity. Our results with rhesus monkeys and a few human donors indicate that peripheral blood mononuclear cells (PBMC) of most uninfected individuals are capable of at least a low-level envelope-specific, UR lytic activity. A fraction of the macaque population had high UR lytic activity, and these animals succumbed more rapidly to AIDS than those with low UR lytic activity. Since UR lytic activity is a property of uninfected PBMC, UR lysis is likely to mediate cell death early in infection and may impair the subsequent immune response to virus.

	MATERIALS AND METHODS

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Virus stocks and cell lines. SIVmac239 and SIVmac251 were filtered, stored, and titered with respect to tissue culture infectious dose (TCID) as described elsewhere (21). Recombinant vaccinia viruses VVwt, VVenv, VVgag, VVpol, and VVnef (gift from Therion Biologics, Cambridge, Mass.) were derived from the NYCBH vaccinia strain and contained no insert or the env, gag, pol, and nef genes from SIVmac251, respectively.

Animal infection and sampling. Fifty-six uninfected monkeys were chosen from the 3- to 8-year age group for our study of UR lytic activity levels. Samples were taken three to five times for each monkey over a 3-year period and tested for levels of UR lytic activity against human B-LCL targets infected with VVenv, VVwt, or VVgag. Eleven of these rhesus macaques were inoculated intravenously with 40 TCID of SIVmac251 as reported previously (5). Animals were confirmed positive for infection by two independent virus isolation assays and two tests for SIV p27 antigenemia. Venous blood samples were collected at 4 weeks before infection or 2, 7, 10, 13, and 16 weeks after infection and then every 4 weeks until euthanasia. All samples were tested to measure p27 plasma antigenemia, virus isolation efficiency (cells and plasma), CD4⁺, CD8⁺, and CD20⁺ lymphocyte subsets, and the UR lytic ability of PBMC against SIV env-expressing target cells. Animals were monitored for clinical status and laboratory measures of disease progression that could be correlated with survival (5).

Construction of plasmids. Three regions of SIVmac239 gp130 were cloned into the mammalian expression vector pcDNA3 (Invitrogen, San Diego, Calif.) to make three constructs: the N-terminal construct (amino acids 1 to 284) pcDNA3/preV3, the central construct (amino acids 285 to 403) pcDNA3/envV3, and the C-terminal construct (amino acids 404 to 1335) pcDNA3/postV3. Oligonucleotide primers used in the construction, made on an automated synthesizer (Gene Assembler Plus; Pharmacia LKB, Piscataway, N.J.), were 5'-AAGGATCCGTAAGTATGGGATGTCTT-3' and 5'-AAGAATTCTCAAAAGCCTGAATA-3' to amplify the N-terminal region, 5'-GGATCCGTAAGTATGCCTAAATGTTC-3' and 5'-GAATTCTCATCCAGTATACCTGG-3' to amplify the central region, and 5'-CGGGATCCGTAAGTATGTGGACAAAT-3' and 5'-GGAATTCTCACAAGAGAGT-3' to amplify the C-terminal region. As a control, the SIV gag gene was also subcloned into pcDNA3, using primers 5'-GGGGTACCTGGGAGATGGGCGTGAGA-3' and 5'-GGAATTCCTACTGGTCTCC-3'. PCR amplification of SIV env and gag genes was performed as described elsewhere (22) with the above primers and plasmids containing SIVmac239 env (SIVMM239spE3') and gag (SIV239spsp5') sequences, respectively. PCR-amplified SIV env fragments digested with BamHI and EcoRI, and SIV gag fragments digested with KpnI and EcoRI, were inserted into pcDNA3 and transfected into Escherichia coli JM109 (Promega, Madison, Wis.), and clones were selected for ampicillin resistance. DNA from resistant clones was purified with the Wizard Plus Minipreps DNA purification system (Promega), screened for insertion of SIV env or gag by restriction enzyme digestion, and further confirmed by DNA sequencing.

Transfection and selection. Human B-LCL were transfected with a pcDNA3 plasmid containing SIV env or gag (pcDNA3/env or pcDNA3/gag, respectively) by electroporation (Gene Pulser; Bio-Rad, Rockville Center, N.Y.) at 0.3 kV and cultured in RPMI 1640-10% fetal calf serum; 48 h after transfection, Geneticin (500 µg/ml; Gibco) was added for selection of neomycin-resistant cells. Two weeks later, RNA was isolated from transfectants by using Trizol reagent (Life Technologies, Gaithersburg, Md.) followed by reverse transcription-PCR to detect expression of the inserted SIV env or gag sequences as described previously (33). The surface expression of SIV env was confirmed by flow cytometry using a monoclonal antibody against SIVmac251 envelope protein (KK46; NIH AIDS Research and Reference Reagent Program, Rockville, Md.).

Preparation of effector cells. Effector PBMC were derived from heparinized whole blood of uninfected people, uninfected rhesus monkeys, or SIV-infected rhesus monkeys by Ficoll-Hypaque (Sigma, St. Louis, Mo.) density gradient centrifugation (37). Polyclonal CD4⁺, CD8⁺, or CD16⁺ lymphocyte subsets were prepared from PBMC by negative or positive selection with immunomagnetic beads (Miltenyi Biotec Inc., Auburn, Calif.). The purity of magnetic bead-selected lymphocyte subsets was checked by flow cytometry. For most experiments, PBMC were stimulated in vitro with concanavalin A (ConA; 5 µg/ml; Sigma) for 3 days followed by 4 days in recombinant interleukin-2 (IL-2; 20 U/ml; gift from John Detrich, Biological Response Modifiers Program, National Cancer Institute, Vienna, Va.). Unstimulated PBMC were used for some experiments. Lymphokine-activated killer (LAK) cells were made by incubating PBMC for 4 days with 500 U of recombinant IL-2 per ml. T-cell clones were a gift from Y. Gan in the laboratory of M. Malkovsky, University of Wisconsin, Madison.

Chromium release assay. Targets were ⁵¹Cr-labeled cells, uninfected or infected with VVwt, VVgag, VVpol, VVnef, or VVenv at a multiplicity of infection (MOI) of 3 PFU per cell for 16 h at 37°C. For some selected assays, cells transfected with pcDNA3/gag or pcDNA3/env were used as targets. Effectors were either total PBMC or lymphocyte subsets selected by magnetic beads. Some assays were performed with ⁵¹Cr-labeled PBMC and unlabeled SIV env-expressing cells. Standard 4- or 5-h chromium release assays were performed in 96-well U-bottom microtiter plates (Costar, Cambridge, Mass.) as described previously (34). Samples were assayed in triplicate. Percent specific lysis was determined as 100 × (experimental release  spontaneous release)/(maximum release  spontaneous release). Maximum release was determined by the lysis of targets in 1% Triton X-100. Spontaneous release was determined by the lysis of targets in medium without effectors and was consistently less than 20%. All assays contained as negative controls uninfected target cells which always showed below 5% specific lysis. Assays were also performed in the presence of different concentrations of EGTA (Sigma), NH₄Cl (Sigma), phenylmethylsulfonyl fluoride (PMSF; Sigma), actinomycin D (Sigma), cycloheximide (Sigma), soluble CD4 (NIH AIDS Research and Reference Reagent Program), monoclonal antibody against CD4 (Antigenix America Inc., Franklin Square, N.Y.), monoclonal antibody against SIVmac251 gp130 (KK46), and monoclonal antibodies against MHC class I (W6/32) and MHC class II (HB180) (gifts from M. Malkovsky). All the above reagents were effective with positive controls and were nontoxic to cells at the maximum specified concentrations.

Statistical analysis. SIV p27 levels, cell counts for lymphocyte subsets, UR lytic ability of PBMC, and weeks of animal survival were analyzed by SAS (Research Triangle, N.C.) JMP statistical software. The survival curve was drawn by using the Kaplan-Meier method. Correlations between any two parameters were tested to identify statistically significant differences.

	RESULTS

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PBMC from uninfected people or rhesus macaques lyse SIV env-expressing cells. Chromium release assays with rhesus macaque PBMC effectors and B-LCL targets at an effector/target (E/T) ratio of 50:1 gave us the initial indication that uninfected PBMC could lyse SIV env-expressing targets (Table 1). B-LCL expressing SIV protein Gag or Nef, after infection with VVgag or VVnef, were not similarly lysed by naive effectors (Fig. 1a). When infected PBMC were used as effectors, MHC-restricted lysis could be distinguished from UR lysis by its sensitivity to EGTA, as shown below. Further CTL assays showed that lysis can occur against several different targets expressing SIV env, e.g., allogeneic rhesus B-LCL (B cells), human B-LCL (B-cell line), CEMx174 (human T-cell/B-cell hybrid), C1R (HLA-A, B null, B cells), and Daudi (HLA-DR⁺, B cells) (Fig. 1b).

View larger version (49K): [in this window] [in a new window]   FIG. 1.   UR lysis is specific for SIV env, occurs in several different targets, and can be initiated by PBMC with or without secondary stimulation. (a) Targets were 51Cr-labeled monkey B-LCL (from monkey 92036) either uninfected or infected with VVwt, VVgag, VVnef, or VVenv. Effectors were allogeneic PBMC from uninfected rhesus macaque (90120) which were stimulated with ConA (5 µg/ml) for 3 days followed by IL-2 (20 U/ml) for 4 days. Targets and effectors were incubated together in a 5-h 51Cr release assay. Results are shown as means ± SEM from three assays. (b) Various targets were infected with VVenv or VVwt. In vitro-stimulated PBMC from an uninfected monkey were used as effectors and were incubated with the targets at an E:T ratio of 50:1 in a 5-h 51Cr release assay in the presence of 1 mM EGTA to block lysis of xenogeneic targets. Results are shown as means ± SEM from three assays. (c) Fresh uninfected (uninf) PBMC (monkey 91070) were cultured overnight to remove adherent cells. Effectors were either whole PBMC (lanes 1 to 3), CD8 cells (lanes 4 to 6), or CD8+ cells (lanes 7 to 9) purified after retention on magnetic bead cell sorting columns. By flow cytometry, the CD8 cells had >82% CD4+ cells and the CD8+ cells had >98% CD8+ cells. A chromium release assay was performed with allogeneic B-LCL targets infected with VVenv. Uninfected and VVpol-infected B-LCL were control targets. E:T ratios were 25:1 (lanes 1, 4, and 7), 50:1 (lanes 2, 5, and 8), and 50:1 with 1 mM EGTA (lanes 3, 6, and 9). * indicates EGTA-resistant lysis. Effector PBMC (stimulated with ConA and IL-2 as described in Materials and Methods) from the same donor were used in parallel (lower panel).

Unstimulated PBMC effectors can cause SIV env-specific UR lysis. PBMC from an uninfected monkey were preincubated on a plastic plate overnight to remove adherent cells and enrich for T cells. In the presence of EGTA, these unstimulated PBMC, specifically the CD8 (CD4⁺-enriched) cells, could mediate UR lysis of SIV env-expressing targets but did not lyse control targets (uninfected cells or cells infected with VVwt). Unstimulated PBMC from two of the other five monkeys tested showed a similar lytic ability (Table 1). UR lytic activity was lower from unstimulated PBMC than from PBMC that had undergone secondary stimulation (Table 1; Fig. 1c).

UR lysis of SIV env-expressing cells is independent of extracellular calcium. Chromium release assays were performed in the presence of EGTA to test whether the UR lysis is sensitive to calcium depletion (Fig. 2). EGTA from 1 to 8 mM failed to inhibit the UR lysis of human B-LCL expressing SIV env, and 8 mM EGTA was not toxic for target cells (Fig. 2a). EGTA at 1 mM blocked lysis of K562 cells by LAK cells (PBMC stimulated with 500 U/ml IL-2 for 4 days) (Fig. 2b), blocked lysis of Daudi cells by T-cell clones (Fig. 2c), blocked lysis by monkey effectors of xenogeneic human targets (Fig. 1b, 3b, 4b, 5, 6c, 7, and 8), and blocked lysis of MHC-restricted targets by antigen-stimulated effectors (data not shown). Thus, 1 mM EGTA was added to most of our assays to ensure that we observed only UR lysis.

View larger version (37K): [in this window] [in a new window]   FIG. 2.   UR lysis of env-expressing target cells is independent of extracellular calcium. (a) Targets were 51Cr-labeled human B-LCL infected with VVenv. Effectors were uninfected allogeneic human PBMC which were stimulated by ConA (5 µg/ml) for 3 days followed by IL-2 (20 U/ml) for 4 days. Targets and effectors were incubated together in a 4-h 51Cr release assay in the absence or presence of different concentrations of EGTA (0 to 8 mM) at an E:T ratio of 50:1. Uninfected B-LCL and B-LCL infected with VVgag were used as negative controls and exhibited less than 5% specific lysis. Results are shown as means ± SEM from three assays. (b) Targets were K562 cells either uninfected or infected with VVwt or VVenv. Effectors were LAK cells from monkey 91070 (left) or one human donor (right). Targets and effectors were incubated in a 4-h 51Cr release assay in the absence or presence of 1 mM EGTA at different E:T ratios. Results are shown as means ± SEM from three assays. (c) Targets were Daudi cells either uninfected or infected with VVwt or VVenv. Effectors were T-cell clone I (left) or clone D (right) from monkey 91035. Targets and effectors were incubated in a 4-h 51Cr release assay in the absence or presence of 1 mM EGTA. Results are shown as means ± SEM from three assays.

UR lysis occurs in VVenv-infected, SIV env-transfected, and SIV-infected cells and can be mediated by the central portion of env. To rule out the possibility that only SIV env from recombinant vaccinia virus can mediate UR lysis, SIV env was additionally expressed from SIV env-transfected cells and from SIV-infected cells (Fig. 3). Only the central portion of env was essential for mediating EGTA-resistant UR lysis (Fig. 3a). To prepare targets infected with whole virus, we infected CEMx174 cells with SIVmac239 at 1 TCID per cell. The SIV-infected CEMx174 cells served as good targets for UR lysis by 2 and 3 days after infection but not after 1 day (Fig. 3b). The occurrence of sensitivity to UR lysis coincided with higher levels of env expression on the cell surface, as shown by flow cytometry (Table 2).

View larger version (18K): [in this window] [in a new window]   FIG. 3.   UR lysis requires only the central portion of the gp120 molecule and occurs in SIV-infected cells after >24 h of infection. (a) Targets were human B-LCL untransfected or transfected with pcDNA3, pcDNA3/gag, pcDNA3/preV3, pcDNA3/envV3, or pcDNA3/postV3. Effectors were human PBMC stimulated by ConA (5 µg/ml) for 3 days followed by IL-2 (20 U/ml) for 4 days. Targets and effectors were incubated together in a 4-h 51Cr release assay. Results are shown as means ± SEM from three assays. (b) Targets were 51Cr-labeled CEMx174 cells uninfected or infected with SIVmac239 at a TCID of 1 for 24 to 72 h or VVgag or VVenv at an MOI of 3 for 16 h. Effectors were PBMC from uninfected rhesus macaque 91070 which were stimulated by ConA (5 µg/ml) for 3 days followed by IL-2 (20 U/ml) for 4 days. Targets and effectors were incubated together in a 5-h 51Cr release assay in the presence of 1 mM EGTA to block lysis of xenogeneic targets. Results are shown as means ± SEM from three assays.

Lysis of SIV env-expressing targets does not require MHC-restricted antigen presentation. To determine whether the env-specific lysis of allogeneic or xenogeneic targets is restricted by MHC, we preincubated the targets with and performed chromium release assays in the presence of monoclonal antibodies against MHC class I (W6/32) and/or MHC class II (HB180). Env-specific lysis by naive effectors could not be blocked by W6/32 (20 µg/ml), by HB180 (20 µg/ml), or by the two combined (Fig. 4a). The above concentration of W6/32 can block MHC-restricted CTL responses (e.g., between rhesus effectors and autologous targets expressing SIV gag); similarly, HB180 can block proliferative responses of rhesus PBMC to Gag antigen p27 (reference 29 and data not shown).

View larger version (30K): [in this window] [in a new window]   FIG. 4.   Lysis of SIV env-expressing targets is not mediated by an MHC-restricted pathway. (a) Lysis of SIV env-expressing targets cannot be blocked by monoclonal antibodies against MHC class I and/or MHC class II. Targets were 51Cr-labeled human B-LCL transfected with pcDNA3/envV3 and preincubated with anti-MHC class I (W6/32, 20 µg/ml) and/or class II (HB 180, 20 µg/ml) for 30 min. Effectors were uninfected human PBMC stimulated by ConA (5 µg/ml) for 3 days followed by IL-2 (20 U/ml) for 4 days. The 4-h 51Cr release assay was done in the absence or presence of anti-MHC class I and/or anti-MHC class II at an E:T ratio of 50:1. pcDNA3/gag-transfected human B-LCL were used as a control. Results are shown as means ± SEM from three assays. (b) Effect of NH4Cl or PMSF on SIV env-specific UR lysis. Targets were human B-LCL infected with VVenv for 2 h and then incubated in the presence of NH4Cl (0 to 20 mM) or PMSF (0 to 20 µg/ml) overnight. Effectors were uninfected monkey PBMC stimulated by ConA (5 µg/ml) for 3 days followed by IL-2 (20 U/ml) for 4 days. The 4-h 51Cr release assay was done in the continuous absence or presence of the above reagents at an E:T ratio of 50:1. Uninfected B-LCL and B-LCL infected with VVgag were used as controls and showed less than 5% specific lysis. EGTA (1 mM) was added to exclude the lysis by monkey PBMC of xenogeneic human targets. Results are shown as means ± SEM from three assays.

SIV env-specific UR lysis does not require de novo transcription or translation in target cells. Target cells were incubated with 0 to 20 µg of actinomycin D, a transcription inhibitor, or of cycloheximide, a protein synthesis inhibitor, per ml for 2 h prior to addition of effector cells in the standard chromium release assay. Neither actinomycin D nor cycloheximide blocked UR lysis at the highest concentrations tested (Fig. 5). Actinomycin D at 5 µg/ml and cycloheximide at 10 µg/ml have been shown to be capable of inhibiting in vitro RNA transcription and protein synthesis, respectively, and both reagents are not toxic to cells at 20 µg/ml (data not shown). Thus, UR lysis was not dependent on de novo transcription or translation.

View larger version (54K): [in this window] [in a new window]   FIG. 5.   SIV env-specific UR lysis does not require de novo transcription or translation in target cells. Targets were 51Cr-labeled human B-LCL pretreated with 10 µg actinomycin D or cycloheximide per ml and infected with VVenv. Effectors were uninfected rhesus monkey PBMC stimulated by ConA (5 µg/ml) for 3 days followed by IL-2 (20 U/ml) for 4 days. The 4-h 51Cr release assay was done in the absence or presence of different concentrations of actinomycin D (Act-D; 0 to 20 µg/ml) or cycloheximide (CHX; 0 to 20 µg/ml) at an E:T ratio of 50:1. Uninfected B-LCL and B-LCL infected with VVgag were used as controls and showed less than 5% specific lysis. EGTA (1 mM) was added to exclude the lysis by monkey PBMC of human targets. Results are shown as means ± SEM from three assays.

CD4⁺ effectors have the most lytic activity. Polyclonal CD4⁺ or CD8⁺ T-lymphocyte subsets were prepared from PBMC by negative or positive selection with immunomagnetic beads. The purity of each subset was tested by flow cytometry. Chromium release assays were performed with five different populations of effectors: (i) total PBMC, (ii) positively selected CD4⁺ cells, (iii) positively selected CD8⁺ cells, (iv) CD4 cells, and (v) CD8 cells. The positively selected populations, in which the column retained cells bound to magnetic beads, were always the most homogeneous (>95% pure by flow cytometry), whereas the depleted populations, comprising cells that failed to bind magnetic beads and flowed through the column, were less homogeneous (>80% pure by flow cytometry, with the CD4 population contaminated by 12% CD4⁺ cells). The majority of the UR lytic activity against SIV env-expressing targets could be accounted for by the CD4⁺-enriched populations (references 7 and 8 and Fig. 6a).

View larger version (45K): [in this window] [in a new window]   FIG. 6.   Lysis of envelope-expressing targets is mediated by CD8 (CD4+) lymphocytes (a) and is less likely related to CD16+ NK activity (b and c). (a) Targets were 51Cr-labeled allogeneic B-LCL infected with VVenv. In vitro-stimulated PBMC were incubated with anti-CD4 or anti-CD8 magnetic beads and separated into four populations: CD4+, CD4, CD8+, and CD8. Flow cytometric analysis was used to determine the purity of the CD4+ and CD8+ populations as greater than 90% and of the CD4 and CD8 populations as greater than 80%. VVgag-infected targets were also tested and gave only baseline lysis (data not shown). Results are shown as means ± SEM from three assays. (b) Stimulated monkey PBMC were depleted of CD16+ lymphocytes by using magnetic bead cell sorting and used to test for lytic activity against SIV env-expressing allogeneic B-LCL at an E:T ratio of 25:1. VVgag-infected targets were also tested as a negative control. Results are shown as means ± SEM from three assays. (c) Targets were 51Cr-labeled K562 cells infected with VVwt or VVenv. In vitro-stimulated monkey PBMC were incubated with anti-CD4 or anti-CD8 magnetic beads and separated into four populations: CD4+, CD4, CD8+, and CD8, as described above. A 4-h 51Cr release assay was performed in the presence of 1 mM EGTA to exclude lysis by monkey PBMC of human targets. Results are shown as means ± SEM from three assays.

SIV env-specific UR lysis may not be mediated by NK cells. Two methods were used to determine whether the SIV env-specific UR lysis is caused by NK cells: we determined whether cells bearing the CD16 marker (an NK marker) were essential for this lysis, and we determined whether classical NK targets, e.g., K562 cells, were lysed in a similar manner. CD16 depletion had no effect on lytic activity (Fig. 6b). K562 cells were lysed only by CD8⁺ lymphocytes, not by CD4⁺ lymphocytes, confirming classical CD8⁺ NK cell activity (Fig. 6c). Also, K562 lysis is sensitive to EGTA, whereas UR lysis is not (Fig. 2 and 6c). Therefore, both tests showed that the SIV env-specific UR lysis is not NK mediated.

UR lysis is blocked completely by antibody to gp130 but only partially by soluble CD4 or antibody to CD4. Chromium release assays were performed with SIV env-expressing targets preincubated with monoclonal antibody against SIV gp130. This treatment at 10 µg/ml could cause an 87% block of the UR lysis and could totally inhibit lysis at 50 µg/ml (Fig. 7a). Assays in the presence of soluble CD4 or effectors pretreated with monoclonal antibody against CD4 showed that soluble CD4 or anti-CD4 treatment could only partially block the UR lysis (Fig. 7b).

View larger version (23K): [in this window] [in a new window]   FIG. 7.   Lysis of SIV env-expressing targets can be inhibited by anti-gp130, soluble CD4, or anti-CD4. (a) VVenv-infected human B-LCL were pretreated with different concentrations of monoclonal antibody against SIVmac251 gp130 for 30 min and incubated with in vitro-stimulated monkey PBMC in a 4-h standard chromium release assay in the presence of anti-gp130. Anti-gp130 at 50 µg/ml was not toxic to cells. Results are shown as means ± SEM from three assays. (b) In vitro-stimulated monkey PBMC were either nontreated or pretreated with monoclonal antibody against CD4 for 30 min and then incubated with VVenv-infected human B-LCL in the presence of soluble CD4 (sCD4) or anti-CD4 in a 4-h standard chromium release assay. Higher concentrations of soluble CD4 and anti-CD4 were also tested and could not increase blockage of the UR lysis. Mouse immunoglobulin G (IgG) was included as an isotype control for anti-CD4. Uninfected B-LCL and B-LCL infected with VVgag were used as controls and showed less than 5% specific lysis. EGTA (1 mM) was added to exclude the lysis by monkey PBMC of human targets. Results are shown as means ± SEM from three assays.

Both targets and effectors are killed. Monkey PBMC stimulated in vitro were depleted of CD8⁺ T cells, labeled with ⁵¹Cr, and then incubated with unlabeled target cells at an E:T ratio of 5:1 for 4, 7, and 18 h. Effector cell lysis was more pronounced when cells were incubated with SIV env-expressing targets than when they were incubated with VVgag-infected targets. Data for 7-h assays are presented because this time point showed the greatest difference for effector cell lysis (Fig. 8). At 4 to 5 h, effector cell lysis was lower and at 18 h there was no significant difference between effector lysis after incubation with different targets.

View larger version (41K): [in this window] [in a new window]   FIG. 8.   CD8 effector cells are also lysed when incubated with SIV env-expressing targets. Uninfected monkey PBMC stimulated in vitro were depleted of CD8+ T cells by using CD8-conjugated magnetic beads and labeled with 51Cr. The labeled cells were then incubated with unlabeled SIV gag- or SIV env-expressing human B-LCL at an E:T ratio of 5:1 for 7 h in the presence of 1 mM EGTA. Supernatant was then collected and counted in a gamma counter. Results are shown as means ± SEM from three assays.

UR lytic activity is prevalent in naive effectors from rhesus macaques. Uninfected 3- to 4-year-old rhesus monkeys, male and female, were screened for the presence of UR lytic activity against VVenv-infected human B-LCL targets in the presence of EGTA. Each animal was tested at least three to five times over a 3-year period, and variation was less than 5% specific lysis per animal from assay to assay. UR lytic activity is shown at an E:T ratio of 50:1, which was maximum activity (plateau) in these assays. Of 56 monkeys, 41% had low UR lytic activity (<15% specific lysis) and 59% had high activity (>15% specific lysis) (Table 3).

High levels of UR lytic activity are negatively correlated with survival. Eleven rhesus monkeys were infected with SIV and monitored. Blood was drawn before and at different time points after infection for laboratory analysis. Euthanasia was performed when an animal showed deterioration of clinical condition, as determined by a veterinarian, usually including weight loss, poor fluid intake, loss of appetite, and unresponsiveness. Simian AIDS was confirmed by both the major pathological diagnosis and the lymphoid tissue status at necropsy (5). Tissue pathology and clinical laboratory data confirmed that euthanasia was appropriate for these animals owing to advanced disease. The ability of PBMC to lyse SIV env-expressing targets at an E:T ratio of 50:1 was used as a standard for comparison among all the monkeys (Fig. 9). Laboratory data before and after infection were correlated with survival to assess the predictive value for each parameter. There was a negative correlation between SIV p27 antigen level and survival which was most significant by 10 weeks postinfection (correlation coefficient 0.6797, P = 0.02) (5). To find the correlation between UR lysis and survival, the 11 monkeys were subdivided into two groups according to the level of UR lysis before infection: those with less than 10% specific lysis and those with greater than 15% specific lysis (Fig. 9a). The survival curve drawn by using Kaplan-Meier method (Fig. 9b) showed a strong negative correlation between the UR lytic ability of monkey PBMC prior to infection and animal survival after SIV infection (P = 0.0368). PBMC from monkeys 2 weeks after infection, but not PBMC from monkeys 10 and 16 weeks after infection, showed similar correlation (data not shown).

View larger version (17K): [in this window] [in a new window]   FIG. 9.   High levels of lytic activity are correlated with rapid disease progression after SIV infection of rhesus monkeys. Eleven rhesus macaques were infected with SIVmac251 at 40 TCID per animal and were monitored for clinical signs of disease progression. PBMC from the 11 monkeys obtained at 4 weeks before infection were cultured in the presence of ConA (5 µg/ml) for 3 days followed by IL-2 (20 U/ml) for 4 days and were tested for the ability to lyse VVenv-infected human B-LCL cells in a 4-h 51Cr release assay. The assay was performed in the presence of 1 mM EGTA to exclude lysis of xenogeneic targets. Uninfected or VVgag-infected targets were also included, and only baseline lysis (below 5%) was obtained. Monkeys were divided into two groups according to the percent specific lysis at an E:T ratio of 50:1: high-lysis group (15%) and low-lysis group (10%) (a). The survival curve for the high-lysis versus low-lysis group was drawn by using the Kaplan-Meier method (b).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Numerous studies described MHC-restricted CTL activity directed toward the HIV or SIV envelope (3, 8, 9, 14, 17, 19, 20, 28, 30-32, 36). Some of these studies reported lysis of env-expressing targets by effector cells from uninfected or unimmunized donors (8, 14, 19, 28, 31). Here we describe UR, SIV env-specific, and EGTA-resistant lysis by effector cells from naive rhesus macaques. These studies confirm the existence of UR cytolysis specific for viral env-expressing targets. The UR lytic activity was studied in samples from more than 50 uninfected monkeys. In a subset of these animals infected with SIVmac, we discovered that the levels of innate UR lytic activity in preinfection samples was directly correlated with the risk for rapid disease progression.

We characterized effector cell phenotype, properties of target cells, and killing mechanisms for the unrestricted, env-specific lysis. Unrestricted lysis could be seen in cultures of fresh, unstimulated lymphocytes and in cultures subjected to secondary stimulation with IL-2. Thus, cytolytic activity is not a consequence of in vitro stimulation. It was reported that UR lysis could be eliminated by stimulating effector lymphocytes with SIV antigen rather than IL-2 (14). This is consistent with the observation that UR lysis eliminates both targets and effectors (references 8 and Fig. 8); i.e., stimulation with Env antigen could eliminate the effectors of UR lysis. Thus, the absence of UR lysis could be the result of the stimulation protocol.

To identify the effectors of UR lysis, we isolated and tested lymphocyte subsets. Magnetic bead depletion of NK cells with anti-CD16 (used by others [31]) failed to subtract the UR lysis. Since not all NK cells express CD16, we performed additional assays using classic NK targets (K562) and showed that there was no preferential lysis of SIV env-expressing K562 cells and that, unlike UR lysis, K562 cell lysis was mediated by CD8⁺ T cells and was EGTA sensitive. Also, UR lysis occurred equally well in both MHC-expressing and -nonexpressing cells, unlike NK lysis. Thus, UR lysis is not mediated by NK cells.

Effector cells for UR lytic activity are CD4⁺, and lysis can be blocked with soluble CD4 or antibody to envelope (references 7 and 8 and this study). Since binding of soluble CD4 or anti-Env inhibits rather than promotes lysis, the CD4-Env interaction is not a death-signalling event, but probably serves to bind cells together and facilitate another mechanism of cell death.

Studies were performed to determine the mechanism of UR lysis. Two distinct molecular pathways for lymphocyte-mediated cell death can be distinguished by their sensitivity to calcium depletion by EGTA (4): the perforin-mediated pathway, which employs calcium-dependent granule-exocytosis and leads to target cell membrane alteration and death, and the Fas/FasL pathway, which transduces apoptotic death signals and is relatively insensitive to calcium depletion by EGTA. UR lysis is insensitive to EGTA (reference 7 and this study) and is therefore similar to Fas-mediated lysis and unlike perforin-mediated lysis. EGTA insensitivity also distinguishes UR lysis from cytolysis by NK cells, T cells, and T cells. Another feature of the Fas pathway that resembles UR lysis is that there is no need for de novo transcription or translation for target cell death (reference 4 and this study). Also, as for the Fas pathway, inhibitors of apoptotic signaling are capable of inhibiting UR lysis (16). Depending on the cell types being considered, different cell surface molecules, e.g., TRAIL (10), Fas (2), and CD4 (1), have been implicated in initiating apoptosis during HIV infection. Although we have not confirmed which of the surface receptors are most important for UR lysis, the mechanism for killing env-expressing cells shares several characteristics with the Fas/FasL apoptotic pathway.

MHC antigen presentation is not involved in UR lysis. Both allogeneic and xenogeneic (human and rhesus) effectors caused SIV Env-specific UR lysis, and lysis was not blocked by inhibitors of MHC antigen processing and presentation. UR lysis cannot be blocked by monoclonal antibodies against common forms of MHC class I and/or class II. It is insensitive to the presence of NH₄Cl, a lysosomotropic agent, and to the presence of PMSF, a proteasome inhibitor. Furthermore, SIV env-expressing C1R cells, which are low in MHC class I expression, and Daudi cells, which are deficient in ₂-microglobulin and express only HLA-DR, are highly susceptible to UR lysis. Thus, UR lysis is not dependent on classical MHC antigen presentation.

To assess the role of SIV env in mediating UR lysis, we monitored surface expression of SIV env by flow cytometry and blocked UR lytic activity with antibody to SIV env. It has long been known that cell surface expression of SIV Env (gp130) can mediate syncytium formation with cells expressing CD4 (24); therefore, it is reasonable to expect a similar mechanism of cell-cell interaction during UR lysis. Similar to CD4-Env-mediated fusion for syncytium formation, UR lysis could be blocked by antibody to SIV Env, and both target and effector cells were killed (7). However few syncytia were observed in cell cultures undergoing UR lysis (reference 7 and this study), possibly because contact between CD4 effectors and env-expressing targets causes cell death so rapidly that syncytium formation is missed.

To assess further the role of SIV env in mediating UR lysis, we expressed truncated forms of SIV env in target cells. The central one-third of SIV Env gp130 could mediate UR lysis. Whereas the central portion of HIV Env gp120 contains a V3 loop, principal CTL epitopes, neutralizing and tropism determinants, and binding sites for chemokine receptors, SIV Env does not contain a well-defined V3 loop and little is known about its function. Both the complete HIV Env and the truncated Env lacking the intracellular portion of gp41 could sensitize cells to UR lysis, although lysis caused by the latter was less pronounced (7). In contrast to findings with HIV Env, we did not need to append a signal sequence to the SIV Env construct to see UR lysis. Similar to findings with HIV Env, sensitivity to UR lysis is correlated with the level of Env expression on infected cells. However, our experiments do not distinguish whether surface or internal Env is essential for mediating UR lysis. Thus, the Env portion could conceivably promote UR lysis from inside the cell, and it is also possible that exogenous Env can be taken up by cells to mediate the lysis of bystander cells.

The wide variety of target cells susceptible to UR lysis raises the possibility that UR lysis affects several cell types in vivo. Most in vitro CTL assays use immortalized B-cell targets, whereas the most prevalent cells infected in vivo are T cells. We confirmed that T-cell targets are susceptible to UR lysis as well. However, it is entirely possible that the UR lytic activity has a major effect on B cells and accounts for the specific deletion of B cells responding to gp120 antigens (18). Massive B-cell depletion has been seen in AIDS wasting (11), and rapid CD4 and B-cell depletion are hallmarks of rapid disease progression (25).

Finally, we studied UR lysis activity levels in a population of uninfected macaques and compared these levels to disease progression rates when some of the animals were used for infection studies. The levels of UR lysis activity varied within a population of rhesus macaques, but values for individual animals were consistent across several independent samples. When 11 of these animals were used for SIVmac infection studies, correlations between UR lysis activity and disease progression emerged. We observed an inverse correlation between survival and SIV p27 levels and a positive correlation between survival and early antibody responses (5). We show here that rapid disease progression was strongly correlated (P = 0.0368) with high levels of env-specific UR lysis in the preinfection samples. One important feature of UR lysis is that target and effector cells (CD4⁺) are both killed in the reaction. Accordingly, UR lysis may be related to the rapid CD4⁺ T-cell decline that is characteristic of SIV-infected macaques showing rapid disease progression.