env-Specific Cytotoxic T Lymphocytes - Journal of Virology - American ...

JOURNAL OF VIROLOGY, June 1993, p. 3409-3417

Vol. 67, No. 6

0022-538X/93/063409-09$02.00/0 Copyright © 1993, American Society for Microbiology

Cross-Reactive Lysis of Human Targets Infected with Prototypic and Clinical Human Immunodeficiency Virus Type 1 (HIV-1) Strains by Murine Anti-HIV-1 IIIB env-Specific Cytotoxic T Lymphocytes SUNIL CHADA,* CATALINE E. DEJESUS, KAY TOWNSEND, WILLIAM T. L. LEE, LISA LAUBE, DOUGLAS J. JOLLY, STEPHEN M. W. CHANG, AND JOHN F. WARNER Departments of Molecular Virology and Immunobiology, Viagene Inc., 11075 Roselle Street, San Diego, California 92121 Received 4 December 1992/Accepted 1 March 1993

To evaluate the ability of murine anti-human immunodeficiency virus type 1 (HIV-1) IIIB env cytotoxic T lymphocytes (CTL) to recognize and lyse HIV-1-infected cells, we have constructed a human cell line (Hu/Dd) expressing both the CD4 receptor and the murine H-2Dd major histocompatibility complex (MHC) class I protein. This cell line can be productively infected with HIV-1 and can also function as a target for murine CD8+, class I MHC-restricted CTL directed against the envelope glycoprotein of HIV-1 IIIB. The ability of BALB/c anti-HIV-1 IIIB env CTL to specifically recognize and lyse Hu/Dd target cells infected with divergent HIV-1 strains was tested by using both prototypic and clinical HIV-1 strains. CTL generated by immunization of mice with syngeneic cells expressing either the native or V3 loop-deleted (AV3) envelope glycoprotein from HIV-1 IIIB were able to recognize and specifically lyse HuIDd target cells infected with the HIV-1 prototypic isolates IIIB, MN, WMJ II, SF2, and CC as well as several HIV-1 clinical isolates. These results demonstrate that CTL determinants for HIV-1 env exist outside the hypervariable V3 region, anti-HIV-1 IIIB env CTL appear to recognize common determinants on diverse HIV-1 strains, and classification of HIV-1 strains based on neutralizing antibody reactivities does not appear to correspond to CTL recognition and lysis. The results suggest that the cell-mediated components of the immune system may have a broader recognition of divergent HIV-1 strains than do the humoral components.

When an individual is infected with human immunodeficiency virus type I (HIV-1), an immune response which appears to suppress viral replication is elicited (5). Nevertheless, the virus persists and debilitates the immune system until eventually the virus overcomes immune surveillance, apparently leading to frank AIDS (2). The mechanism by which HIV degrades the immune system is not well understood. If strategies can be found to augment natural immunity against HIV-1, such strategies may allow the development of effective treatment regimens that could alter the normally fatal course of AIDS. Much effort has now been focused on understanding and enhancing the cellular immune response to combat HIV-1 infection. In particular, the induction of CD8+ cytotoxic T-lymphocyte (CTL) responses may be important in preventing HIV-1 spread by destroying virally infected cells and limiting disease progression (42). Vigorous CTL responses have been shown to be an essential component in protection against a number of viral pathogens. CD8+, class I major histocompatibility complex (MHC)-restricted CTL are crucial in the protection of mice against lethal challenge by lymphocytic choriomeningitis virus (4), influenza virus (24), and murine cytomegalovirus (CMV) (34). Depletion of CD8+ T cells prior to virus challenge generally leads to rapid viremia, increased disease severity, and lethality. Adoptive transfer of CD8+ CTL after virus infection can reverse disease progression in animal models (44). *

We have developed a system to elicit consistent CTL responses that uses retroviral vectors as vehicles for gene transfer and antigen presentation. We have shown that retroviral transduction of the HIV-1 envelope (env) gene into murine cells is a potent method for inducing immune responses directed against HIV-1 env-expressing cells (43). Injection of HIV-1 env-expressing murine cells into syngeneic BALB/c mice results in the induction of both vigorous CTL responses and humoral immune responses with virus neutralizing activity. The CTL were shown to be CD8+ and class I H-2Dd MHC restricted. Because murine cells are not infectible by HIV-1, CTL responses were assessed by using target cells coated with peptides derived from the HIV-1 env protein sequence and target cells transduced with a retroviral vector encoding HIV-1 IIIB env. Here we describe the construction of a human cell line that can be productively infected with HIV-1 and expresses the murine H-2D" MHC molecule and therefore can function as a target for murine HIV-1-specific CD8+, class I-restricted CTL. This cell line should facilitate the experimental analysis of CTL responses directed against HIV-1-encoded proteins in an easily manipulated inbred animal system that uses HIV-1-infected cells as targets.

MATERIALS AND METHODS Materials and cells. BALB/c (H-2d) mice were purchased from the Charles River Laboratories, Wilmington, Mass., and Harlan Sprague-Dawley, Indianapolis, Ind. Female mice (6 to 8 weeks old) were used. The murine fibroblast cell line

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BC1OME (BC; H-2d [30]) and CD4+ human cervical carcinoma cell line HeLa-T4+ (25) were grown in Dulbecco modified Eagle medium-10% fetal calf serum (FBS). BC cells were transduced with retroviral vectors, and cloned transduced cells were used for immunization of mice. Cells transduced with and expressing the vector carrying HIV-1 env are designated as env preceded by the abbreviated cell name (e.g., BC-env). The human T-cell lines H9 (33), MT2 (16), and SupTl (17), used for propagation of HIV-1 strains and for detection of HIV-1 envelope expression and infectivity by syncytium formation (see below), were grown in RPMI 1640 supplemented with 20% fetal calf serum. Propagation and titering of HIV-1 strains and vaccinia virus vectors. HIV-1 strains IIIB (33), MN (10), SF2 (22), CC (11), and HIV-2cBL20 (37) were obtained from the AIDS Research and Reference Reagent Program. The infectious chimeric WMJ II strain, obtained from D. Looney (University of California, San Diego), was generated by Beatrice Hahn by replacing the env gene from WMJ II (13) with the env gene of pHXB2D. The clinical isolates were gifts from A. Langlois (patient 1, DU6587-2) and D. Richman (patient 2, G762-3; patient 3, I227-3). All prototypic HIV-1 strains were propagated in H9 cells, whereas the clinical strains were grown in either H9 or MT2 cells, and aliquots of high-titer virus stock were frozen at -80°C. The in vitro 50% tissue culture infectious dose (TCID50) per milliliter for each virus stock on MT2 cells was determined by endpoint dilution analysis (1). Identities of the HIV-1 IIIB and MN strains were confirmed by Western immunoblot analysis using type-specific antisera (data not shown) and by neutralizing antibody reactivity (performed by T. Matthews). The vaccinia virus vectors vSC60 (VAC-GP160) and VAC-LACZ were obtained from B. Moss and D. Kuritzkes, respectively. To prepare stocks of the recombinant vaccinia virus vectors, HeLa S3 cells were infected (multiplicity of infection [MOI] of 1) with the vaccinia virus vectors for 72 h. The cells were then pelleted and lysed with an equal volume of 2.5% trypsin at 37°C for 60 min, and the cell lysate was clarified at 250 x g for 20 min. Virus was first pelleted at 19,000 rpm (type 19 rotor) for 90 min at 5°C and then banded over a 20 to 40% (wt/vol) sucrose gradient at 35,000 rpm (Beckman type 35 rotor) for 2 h. The virus band was recovered from the 20/40% sucrose interface and pelleted for 1 h at 35,000 rpm in a type 35 rotor. The 1,000-foldconcentrated virus was resuspended in phosphate-buffered saline (PBS)-2% FBS as a stock of 109 TCID50 units/ml (titered by endpoint dilution on BSC-1 cells and read at 72 h). Vector constructs and transduction. The retroviral vector construct N2 IIIBenv was generated by inserting the 3.1-kb XhoI-ClaI fragment from the pAF env retroviral provector (containing the HIV-1 IIIB env and rev genes [43]) into the backbone of an engineered N2 murine recombinant retrovirus. For this N2 retroviral construct, the ATG initiator codon for Moloney murine leukemia virus gag was converted to ATT by site-directed mutagenesis (20), resulting in an increased level of expression of transduced genes. For selection purposes, the neomycin phosphoryltransferase (Neor) gene, conferring resistance to the antibiotic geneticin (G418; GIBCO, Grand Island, N.Y.), was included in the vector.

N2 IIIBenv AV3 was derived from N2 IIIBenv and contains the entire HIV-1 IIIB env gene except for an in-frame deletion of 108 bp encompassing the immunodominant hypervariable V3 loop region (amino acids 303 to 338 encompassing the cysteine-bridged loop). A 602-bp HincII-ScaI fragment containing the V3 loop sequences of HIV-1 IIIB env was inserted into plasmid pIBI30 (IBI Inc., New Haven,

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Conn.). A 43-bp oligomer having the sequence 5'-G AAC CAA TCT GTA GAA ATT AAT AAC AAT AGT AGA GCA AAA TGG-3' complementary to the DNA sequence flanking the cysteine bridge of the V3 loop region of HIV-1 IIIB env was synthesized. Site-directed mutagenesis of the mismatched primer was done by the method of Kunkel (20) to generate an in-frame deletion of 108 bp representing the entire V3 loop. This was confirmed by DNA sequencing. The StuI-MstlI fragment containing the V3 deletion replaced the corresponding wild-type sequence in the Bluescript SK+ vector (Stratagene, San Diego, Calif.) containing the HIV-1 IIIB env and rev genes. The XhoI-Clal fragment from this plasmid was inserted into the corresponding sites of the N2 retroviral vector. The SV2neo gene was then inserted into the ClaI site in the sense orientation. The N2 IIIBenv and N2 IIIBenv AV3 provector constructs were transiently transfected into the PA317 amphotropic packaging cell line (27); 24 h later, culture supernatant containing replication-incompetent vector particles was used to transduce the BC cell line at an MOI of 0.1. Cells were treated with G418 (800 mg/ml) for 2 weeks, and G418r colonies were analyzed for HIV-1 env expression (see below). Retroviral vector preparations and supernatant from vector-transduced cells were tested for the presence of replication-competent virus by S+L- assay (31) and scored negative. The H-2Dd expression vector pCMV-Dd hygro was constructed by ligating the 6-kb BamHI-XbaI fragment from c49.2 (a cosmid clone containing the H-2Dd gene [36]) downstream from the human CMV immediate-early (IE) promoter present within the Bluescript SK+ plasmid. This construct utilizes the natural H-2Dd gene poly(A) signal. The hygromycin resistance marker driven by the Moloney murine sarcoma virus promoter (obtained from pY3 [3]) was cloned as a 2.3-kb BamHI-HindIII fragment upstream of the H-2Dd gene. Expression analysis. HIV-1 env-expressing cells (BC-env and BC-envAV3) were tested for expression by Western analysis. Individual clones were lysed, and 20 ,ug of cellular protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted onto polyvinylidene difluoride membranes (Immobilon-P; Millipore) according to the manufacturer's instructions. The anti-gpl20 monoclonal antibody lCl (1:1,000 dilution; Repligen Corp.) was used to probe the blot, and signal was visualized by using 125I-protein A. Expression of H-2Dd was analyzed by Western analysis as described above, using the antibody 7.2.1.4 (1:250 dilution; obtained from R. Dutton), and signal was visualized by using 125I-protein A. For cell surface analysis, expression of H-2Dd was analyzed by either flow cytometry or immunofluorescence, using the monoclonal antibody 34-5-8S (1:100 dilution; a gift from R. Dutton), and visualized by using fluorescein isothiocyanate (FITC)-conjugated rabbit antimouse immunoglobulin G (1:500 dilution; Cappel). For analysis of CD4 expression, OKT4 (1:20 dilution; Ortho Pharmaceutical) was used as the primary antibody and FITCconjugated rabbit anti-mouse immunoglobulin G (1:500 dilution, Cappel) was used as the secondary antibody. For flow cytometric analysis, Hu/Dd cells were treated with trypsin (5 min at 37°C), washed in PBS-5% FBS, and resuspended at 107 cells per ml. Cells (5 x 106) were pelleted through a cushion of FBS, resuspended in 0.5 ml of diluted antibody solution (PBS-5% FBS), and incubated for 45 min at 4°C. The cells were washed twice by pelleting through a cushion of FBS as described above and resuspended in 0.5 ml of diluted FITC-conjugated rabbit anti-mouse secondary

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LYSIS OF HIV-INFECTED HUMAN TARGETS BY MURINE CTL

antibody. After incubation for 45 min at 4°C, cells were washed as described above, resuspended in PBS, and analyzed by flow cytometry (Becton Dickinson FACStar Plus) or immunofluorescence (Nikon Optifot). Construction of the HuIDd cell line. HeLa-T4+ cells (106) were transfected with 10 ,ug of pCMV-Dd hygro by the calcium phosphate procedure and selected in hygromycin (150 mg/ml) for 2 weeks. Antibiotic-resistant cells were treated with 34-5-8S (anti-Dd) primary antibody, a fluorescent labeled secondary antibody, and cells expressing high levels of cell surface H-2Dd were sterilely selected by flow cytometry (Becton Dickinson FACStar Plus). The flowsorted cells were cloned by limiting dilution, and one clone that expressed high levels of H-2D' was selected. This clone was termed Hu/Dd. The Hu/Dd cell line was demonstrated to be free of amphotropic helper virus contamination by S+Lanalysis performed on supernatant obtained from Hu/Dd cells. The Hu/Dd cell line was also shown not to be blocked for infection by amphotropic retrovirus (data not shown). Cytotoxicity assay. BALB/c mice were injected once intraperitoneally with 5 x 106 irradiated (10,000 rads; 'Co) vector-transduced cells (e.g., BC-env). Animals were sacrificed 7 days later, and the splenocytes (3 x 106/ml) were cultured in vitro with irradiated syngeneic transduced cells (6 x 104/ml) in flasks (T-25; Coming Glass Works, Corning, N.Y.). Culture medium consisted of RPMI 1640, heatinactivated FBS (5%; HyClone, Logan, Utah), sodium pyruvate (1 mM), gentamicin (50 mg/ml), and 2-mercaptoethanol (10- M; Sigma Chemical, St. Louis, Mo.). Effector cells were harvested 4 to 7 days later and tested at various effector/target cell ratios in 96-well microtiter plates (Corning) in a standard 4- to 6-h assay. The assay employed Na251CrO4 (Amersham, Arlington Heights, Ill.)-labeled (100 ,uCi; 1 h at 37°C) target cells (104 cells per well) in a final volume of 200 pl (36). Following incubation, 100 ,ul of culture medium was removed and triplicate samples were analyzed in a Beckman gamma spectrometer. Spontaneous release (SR) was determined as counts per minute from targets plus medium, and maximum release (MR) was determined as counts per minute from targets plus 1 M HCl. Percent specific lysis was calculated as [(effector cell + target cpm) - SR/MR - SR] x 100. Spontaneous release values of targets were typically 20% of the MR. Representative results of CTL assays are shown. For generation of HIV-infected targets for CTL assays, Hu/Dd cells were infected with HIV-1 or HIV-2 at a nominal MOI of 0.1. After the infection was allowed to proceed for 8 to 16 h, the plates were rinsed twice and fresh medium was added. Incubation was continued until cytopathic effect (CPE) was evident (7 to 10 days), at which time the cells were washed and removed from plates. The cells were labeled with 51Cr as described above. For generation of recombinant vaccinia virus vectorinfected targets, 5 x 105 BC cells were infected with the recombinant vaccinia viruses at a nominal MOI of 2 at 37°C. After 30 min, the cells were washed and incubated for 16 h. The cells were detached from plates by treatment with EDTA and labeled with 51Cr as described above. RESULTS Retroviral vector-mediated expression of HIV-1 IIIB env. A retroviral provector containing HIV-1 IIIB env (N2 IIIBenv; Fig. la) was constructed by using a modified Moloney murine leukemia virus backbone. Because retroviral vectors are reverse transcribed into DNA and integrate into the host genome, the rev gene of HIV-1 IIIB was provided, as

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FIG. 1. Schematic of recombinant vectors and subsequent HIV-1 env expression. (a) Recombinant retroviral constructs used to express HIV-1 IIIB env and HIV-1 IIIB env AV3. The deletion of the V3 hypervariable loop is indicated. The pCMV-Dd hygro vector used to express the murine H-2D" gene is shown. LTR, long terminal repeat; Prom., promoter; a.a., amino acids; MoMSV,

Moloney murine sarcoma virus. (b) Western blot analysis of lysates from clones of BC cells transduced with the N2 IIIBenv (BC-env) or N2 IIIBenv AV3 (BC-envAV3) retroviral vector. Samples were subjected to SDS-PAGE (8% acrylamide), blotted onto Immobilon-P membranes, and treated with anti-gp12O monoclonal antibody lCl. Signal was visualized by treating the blot with "~I-protein A and autoradiography. Lane 1 shows the rgpl2O standard (ABT).

previously described, to facilitate envelope protein expression (14, 43). The retroviral provector DNA construct was transiently transfected into the amphotropic packaging cell line PA317, and nonreplicating retroviral vectors were generated. N2 IIIBenv retroviral vector particles were used to transduce BC cells (H-2d1), and stable HIV-1 env-expressing clones were isolated. Expression of the HIV-1 TuIB env glycoproteins was assessed in these clonal cell lines by Western blot analysis; the results for one such cell line, BC-env, are shown in Fig. lb.

CTL induction with use of retroviral vector-transduced cells. BC-env cells were injected into syngeneic BALB/c

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FIG. 2. CTLenv-mediated lysis of murine target cells expressing the envelope glycoprotein gene of HIV-1 IIIB. Murine CTLenv were generated by injection of syngeneic BC-env cells (BC cells transduced with the N2 IIIBenv retroviral vector) into BALB/c mice. Splenocytes were removed and restimulated in vitro for 5 to 7 days, and the resulting CTL were tested in a standard 51Cr release CTL assay. Targets tested were BC, BC-env, BC-envAV3, BC cells coated with the RP135 (IIIB-specific V3 loop) peptide, or BC cells infected with vaccinia virus-lacZ or vaccinia virus-gpl60 to generate BC-VAC-LACZ or BC-VAC-GP160, respectively. Spontaneous release values were less than 15%; standard errors of the means of triplicate cultures were less than 7% of the mean.

mice, and env-specific CTL effector cells (CTLenv) were induced. These CTLenv lysed BC-env target cells and appeared to be specific for HIV-1 env protein determinants, as the parental BC cells were not lysed (Fig. 2). However, the recombinant retrovirus N2 IIIBenv, used to transduce HIV-1 env into BC cells, contains the HIV-1 IIIB env and rev genes as well as the bacterial Neor marker. Therefore, to demonstrate that CTLenv were induced, target cells were generated by infection of BC cells with a recombinant vaccinia virus expressing the env gene product of HIV-1 IIIB (BC-VAC-GP160). The vaccinia virus recombinant does not express either the rev gene of HIV-1 or Neor. Figure 2 shows that murine CTLenv generated by injection of BC-env cells into BALB/c mice effectively lysed BC-VAC-GP160 cells and therefore are specific for determinants of the HIV-1 env protein. Furthermore, CTLenv also lysed BC target cells coated with the IIIBenv-specific V3 loop peptide (BCRP135), indicating that the effector population does contain CTL directed against the env V3 loop region (Fig. 2). Development of the Hu/Dd cell line. To evaluate the ability of CTLenv to lyse HIV-1-infected targets, an HIV-1-infectible target was required because murine cells cannot be infected by HIV-1. We therefore transferred the previously identified major HIV-1 env-specific murine class I MHC gene (H-2Dd) into human cells to provide the appropriate antigen presentation components to the HIV-infectible human cells to allow their recognition and lysis by murine CTLenv. We expected that the murine class I protein would be complemented by human P2-microglobulin and immune accessory molecules to produce a hybrid system in which a

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FIG. 3. Lysis of human cells expressing H-2Dd and CD4 and infected with HIV-1. Murine CTLenv were incubated in a 51Cr release assay with HIV-1 IIIB-infected Hu/Dd cells, uninfected Hu/Dd cells, HIV-1 IIIB-infected HeLa-T4+ cells, and uninfected HeLa-T4+ cells as targets. Expression of either HIV-1 env or H-2Dd alone was not sufficient to render targets lysable by CTLenv; coexpression of HIV-1 env and H-2Dd was required for target cell lysis. Spontaneous release values were less than 20%; standard errors of the means of triplicate cultures were less than 5% of the mean.

human cell could present antigen to murine CTL. The HIV-infectible HeLa-T4+ cell line (25) was chosen as a recipient for the murine H-2Dd gene, thereby generating a cell line that could serve as a host for HIV-1 replication and simultaneously function as a target for BALB/c (H-2d) murine CTLenv. The HeLa-T4+ cell line was transfected with pCMV-Dd hygro (Fig. la) and sorted by flow cytometry to generate an H-2Dd-positive cell line (Hu/Dd). Flow cytometric analysis indicated that the Hu/Dd cell line expressed higher levels of cell surface H-2Dd than did the murine BC cell line (data not shown). Infection of Hu/Dd cells by HIV-1 IIIB and lysis by CTIenv. The Hu/Dd cell line was infected with a cell-free HIV-1 IIIB virus preparation (33), and the culture was monitored for CPE and release of p24&a& antigen into the culture medium. HIV-1 virus replication was evident in the Hu/Dd cell line because substantial CPE was observed in the culture within 1 week postinfection and continued for a period of several weeks after infection. Release of p24 antigen into the culture medium increased with CPE for up to 3 weeks after infection (data not shown). The HIV-1 IIIB-infected Hu/Dd cells were used as targets in a "Cr release assay using HIV CTLenv. The HIV-1infected Hu/Dd target cell line was recognized and lysed substantially by CT[env (Fig. 3). HIV-1-infected HeLa-T4+ cells were not lysed, indicating the requirement for the H-2Dd restriction element. Uninfected Hu/Dd cells were not lysed, showing the need for expression of HIV-1 env for recognition by CTLenv. However, when H-2D" and HIV-1 env were coexpressed, the targets were specifically lysed by CTIenv. In control experiments, HIV-1 IIIB-infected Hu/Dd cells were not lysed by murine CTL directed against either the simian immunodeficiency (SIV) envelope glycoprotein or the CMV pp89 IE phosphoprotein (data not shown). Furthermore, CTLenv did not lyse HIV-2-infected (Fig. 4a) or SIV-infected (data not shown) Hu/Dd cells,

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FIG. 4. (a) Lysis of Hu/Dd cells infected with prototypic HIV-1 isolates by murine CTLenv. Hu/Dd cells (106) were infected with different strains of cell-free HIV-1 or HIV-2 (105 TCID50 obtained from chronically infected H9 cells). Ten days postinfection, HIV infected cells were used as targets in a 5-h 51Cr release assay using murine CTLenv. (b) Lysis of Hu/Dd cells infected with clinical HIV-1 isolates by murine CTLenv. Hu/Dd cells (106) were infected with three different patient strains of cell free HIV-1 (Pt.1, Pt.2, and Pt.3; 10' TCID50 obtained from chronically infected MT2 cells). Ten days postinfection, HIV-1-infected cells were used as targets in a 5-h 51Cr release assay using the same murine CTLenv as used for panel a. Spontaneous release values were less than 25%; standard errors of the means of triplicate cultures were less than 4% of the mean.

HIV-1 IIIB-infected H9 cells, or HIV-1 IIIB-infected MT2 cells (the T-cell lines used to propagate the HIV-1 strains; data not shown). These results demonstrated the class I MHC restriction and HIV-1 env specificity of CTLenv. The time course of HIV-1 infection in target cells was investigated. The Hu/Dd cell line was infected with HIV-1 IIIB, and lysis by CTLenv was monitored as a function of time. An increase in specific lysis of the HIV-1-infected cells was observed within 1 week after infection, and substantial levels of lysis (more than 30% specific lysis above the level of uninfected control targets) were observed for a further 2 weeks, during which time CPE was evident. The time course of HIV-1 replication, as monitored by p24 antigen release into the culture medium, correlated with the lysis of HIV-1

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IIIB-infected Hu/Dd targets by murine CTLenv (data not shown). We have previously shown that murine target cells expressing the HIV-1 IIIB envelope gene (generated by retroviral transduction) are lysed by MHC class I-restricted CD8+ CTL (43). CTLenv generated by injection of vaccinia virus-gpl60 into BALB/c mice have also been shown to be of the CD8+ phenotype (38). The phenotype of the effector cells which lysed HIV-1-infected targets was established by depletion of CD8+ cells from the murine CTLenv effector population by using specific monoclonal antibodies and complement. CD8+-depleted effector cells did not lyse either the human targets infected with HIV-1 IIIB or the syngeneic murine BC-env targets (data not shown). Thus, in the CTLenv effector population, only the CD8+ cells appear to lyse the HIV-1-infected target cells. Cross-reactive lysis of cells infected with prototypic and clinical HIV-1 strains by CTIenv. Sequence analyses of V3 loop regions and neutralizing antibody assays have indicated that MN-type viruses represent 60 to 70% of clinical HIV-1 isolates in the U.S. patient population, while IIIB-type viruses represent only 7 to 14% (21). The HIV-1 IIIB and MN strains share approximately 80% sequence similarity in their envelope sequences (12). The ability of CrLenv to specifically recognize and lyse Hu/Dd cells infected with divergent HIV-1 strains was tested by using the prototypic HIV-1 strains IIIB, MN, WMJ II, SF2, and CC and untyped clinical isolates obtained from three HIV-1-infected patients. Hu/Dd cells infected with HIV-2cBL2o were included to control for nonspecific lysis of HIV-infected target cells. Hu/Dd cells were infected with the various HIV-1 and HIV-2 strains, and when significant CPE was evident (approximately 10 days postinfection), the infected cells were used as targets in a 51Cr release assay using murine CTLenv (Fig. 4). CTLenv exhibited substantial lysis of target cells infected with HIV-1 IIIB. Hu/Dd cells infected with HIV-1 MN were lysed by CTLenv less well than with IIIB-infected targets. HIV-1 WMJ II-, SF2-, and CC-infected targets were also lysed by CTLenv, although at lower levels than were HIV-1 IIIB-infected targets (Fig. 4a). Target cells infected with HIV-2 were not lysed by CTLenv. Lysis of HIV-1 MN-, CC-, and patient 1 isolate-infected Hu/D targets was shown to be dependent on CD8+ T cells (data not shown). The clinical strains obtained from three patients also exhibited substantial lysis by CTLenv, although the level of lysis was lower than that observed for IIIB-infected cells (Fig. 4b). The pattern of CTL cross-reactivity was consistent over a period of several weeks after HIV-1 infection of the Hu/Dfd cells. The Hu/D' target cells, infected with the divergent HIV isolates, all exhibited substantial levels of env expression by Western blot analysis (data not shown). Therefore, CTLenv were able to recognize and specifically lyse targets infected with a number of divergent prototypic and clinical HIV-1 strains. These data suggest that the envelope glycoproteins from divergent HIV-1 isolates, including patient isolates, share a common epitope or epitopes that can be recognized by the murine CTLenv population. The CTL cross-reactivity observed with the Hu/Dd cell line is not limited to the HeLa-T4+ cell line system. In parallel experiments, the human H9 and SupTl T-cell lines were transduced with a retroviral vector encoding the H-2Dd gene to generate H9-Dd and SupTl-Dd, respectively. When H9-Dd or SupTl-Dd target cells were infected with divergent HIV-1 strains, substantial CTL cross-reactivity was observed with CTLenv and CTLenvAV3 (4a). Inhibition of HIV-1 IUB replication by murine CTLenv. As another assay of the biological function of CTL, we deter-

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FIG. 5. Inhibition of HIV-1 replication by murine CT'Lenv. Hu/Dd cells were infected as described for Fig. 4 with 105 TCID50 of HIV-1 IIIB. Four days later, the HIV-infected cells were washed and CTLenv were added at the indicated ratios. After 2 weeks in culture, supernatant was removed and the titer of infectious HIV-1 was assessed by endpoint dilution in an MT2 cell syncytium assay.

mined whether CTLenv could diminish HIV-1 replication in infected cultures. HIV-1 IIIB-infected Hu/Dd cells were mixed with CTLenv at various ratios, and the culture was incubated for 2 weeks. Culture supernatant was then removed and tested for infectious HIV-1 IIIB in a syncytium assay. After 2 weeks in culture, a reduction in HIV-1 IIIB titer of greater than 3 logs was observed with addition of the highest number of CTLenv effectors (Fig. 5). At lower input levels of CTLenv cells, reduced levels of inhibition of HIV-1 replication were found, although at an effector/target cell ratio of 10:1, a 50-fold reduction in HIV-1 titer was observed, indicating the ability of murine CTLenv to inhibit HIV-1 IIIB growth in tissue culture. Inhibition of HIV-1 replication by CTLenv was also observed in cells infected with HIV-1 MN (data not shown). Generation of CTL specific for a deletion mutant of HIV-1 IIIB env. It has been reported that the V3 hypervariable loop (amino acids 303 to 338) of HIV-1 IIIB env contains an immunodominant CTL epitope, and this epitope has been further mapped to a 15-amino-acid peptide (38). To examine the relative contribution of the HIV-1 env V3 loop to CTL reactivity, we generated a retroviral construct in which the V3 loop, encompassing the entire cysteine-to-cysteinebridged loop (amino acids 303 to 338), was deleted (N2 IIIBenv AV3; Fig. la). Retroviral vector particles were generated and used to transduce the murine BC cell line, and stable HIV-1 env-expressing clones were isolated. Expression of the HIV-1 IIIB env glycoproteins was assessed in a clonal cell line, BC-envAV3 (N2 IIIBenv AV3 transduced), by Western blotting (Fig. lb). The fully processed gpl60 and gpl20 polypeptides were observed in BC-env cells, whereas BC-envAV3 cells expressed the predicted smaller forms of both proteins. CTLenv lysed BC-envAV3 target cells, although at lower levels than lysis of BC-env (Fig. 2). BALB/c mice were injected intraperitoneally with BCenvAV3 cells, the splenocytes were isolated and restimulated in vitro, and the resulting effector cells were tested for the ability to lyse syngeneic HIV-1 env-expressing target cells. CTL generated by immunization of mice with BCenvAV3 cells (CTLenvAV3) specifically killed BC-envAV3

and BC-env targets (Fig. 6a) but failed to lyse BC target cells coated with the IIIB-specific V3 loop peptide, RP135 (amino acids 307 to 330). CTLenvAV3 specifically lysed BC target cells infected with the vaccinia virus-gpl60 vector (Fig. 6a). These data demonstrate that CTLenvAV3 effectors recognized HIV-1 IIIB env epitopes other than the V3 loop region epitope. To examine the relative contribution of the V3 loop CTL determinants in recognition of divergent HIV-1 isolates, CTLenvAV3 were tested against HIV-1-infected Hu/Dd targets (Fig. 6b and c). CTLenvAV3 lysed HIV-1 IIIB-infected Hu/Dd targets at levels comparable to lysis of BC-env or BC-VAC-GP160, indicating that HIV-1 IIIB-infected human cells display CTL epitopes in association with H-2Dd. Although the V3 loop determinant has been described as an immunodominant CTL epitope in HIV-1 env (41), it is clear that the non-V3 loop CTL determinant(s) contributes substantially to lysis of HIV-1 IIIB-infected human cells. CTLenvAV3 lysed MN-infected Hu/Dd cells at levels comparable to lysis of IIIB-infected target cells. CTLenvAV3 also lysed the divergent prototypic HIV-1 strains WMJ II and CC, although at lower levels than lysis of MN-infected target cells. HIV-2-infected target cells were not lysed by CTLenvAV3. Targets infected with the clinical HIV-1 strains were also lysed substantially by CTLenvAV3 (Fig. 6c). The broad, group-specific CTL cross-reactivity between divergent HIV-1 strains observed with CTLenv is qualitatively similar to the response with CTLenvAV3. However, relative lysis of targets infected with certain HIV strains, most notably MN and the patient 2 isolate, was higher by CTLenvAV3 than CTLenv. Therefore the cross-reactivity found in the current studies is likely due to conserved epitopes found outside the V3 region of HIV-1 env. DISCUSSION In this study, we have generated a human cell line, Hu/Dd, containing both CD4 (the receptor for HIV-1 infection) and the murine class I H-2Dd restriction element (for recognition by murine CTL). The Hu/Dd cell line serves as a novel tool for the analysis of murine CTL responses directed against the HIV-1 envelope glycoprotein and has facilitated an examination of the level of CTL cross-reactivity between divergent HIV-1 isolates. We have used this system to demonstrate that CTLenv can recognize determinants presented by HIV-1-infected cells. The Hu/Dd system has allowed us to evaluate cross-reactivity between CTL induced by immunization with cells transduced with a recombinant retrovirus encoding HIV-1 IIIB env and target cells infected with a number of prototypic and clinical HIV-1 strains. We have shown that envelope determinants from a number of divergent HIV-1 strains are presented on these targets to murine CD8+, MHC class I-restricted HIV-1 env-specific CTL. Experiments using a recombinant retroviral vector lacking the V3 loop of HIV-1 env have shown that CTL reactive against non-V3 region determinants mediate lysis of HIV IIIB env-expressing target cells in addition to cells infected with divergent HIV-1 isolates. All HIV-1infected Hu/Dd cell lines tested were lysed by murine CTLenv and CTLenvAV3, indicating that the H_2Dd MHC molecule is capable of presenting multiple (at least two) oligopeptides from HIV-1 env. CTL cross-reactivity did not extend to Hu/Dd target cells infected with HIV-2 or SIV viral isolates, indicating the specificity of the effectors for HIV-1 env proteins. Previous studies (2a) have demonstrated murine CTL

VOL. 67, 1993 a

100-


Effectors: CTLenv AV3

activity against the T-antigen expressed in simian virus 40-infected monkey cells engineered to express the murine H-2Dd and H-2K' molecules. In a manner similar to that found for the Hu/Dd cell line, these engineered monkey cells were capable of processing and presenting antigen in the context of mouse MHC molecules. These finding suggest the similarity of antigen processing/presentation mechanisms within mammalian cells. Variation in HIV-1 strains is an important factor to consider in the design of specific antiviral therapies and vaccines. It has been well established that HIV-1 env and other proteins vary in amino acid sequence, even during the asymptomatic phase of HIV-1 infection (26). One potential function of this variation in vivo could be to evade the host immune responses, both humoral and cellular. Variations in humoral immune responses directed against HIV-1 env and their correlation with HIV-1 nucleic acid sequence have been demonstrated (21). A classification system for divergent HIV-1 isolates that uses serologic reactivity and V3 loop amino acid sequence analyses has been proposed (18). The use of HIV-1 IIIB-based immunotherapeutic agents may be viewed critically because IIIB does not represent a predominant isolate type, although it was the first available molecularly characterized HIV-1 isolate. The data presented here suggest that although HIV-1 IIIB-type isolates may exist infrequently in the clinical population, significant recognition and cross-reactivity to HIV-1 MN and other divergent strains occurs at the level of the CTL response induced by the envelope glycoprotein of HIV-1 IIIB, as demonstrated in this murine model. The extent of the observed CTL cross-reactivity suggests that one or more conserved, group-specific CTL epitopes may exist in the HIV-1 envelope protein. The HIV-1 env protein contains multiple variable and conserved regions and also multiple CTL epitopes (23). It is therefore not surprising that CTL cross-reactivity was observed and may be attributable to conserved common epitopes. This study has examined eight different HIV-1 prototypic and clinical strains. With this murine model system, no obvious classes of HIV-1-specific CTL reactivity could be defined, quite distinct from the type-specific classification of HIV-1 strains obtained by using principal neutralizing determinant (PND)-specific neutralizing antibody reactivity. HIV-1-specific CTh. Recent studies have demonstrated that murine CTL generated by using vaccinia virus-MN env could kill syngeneic cells coated with V3 peptides from 4 of 14 different HIV-1 strains (40). In addition, when vaccinia virus-IIIB env-primed effectors were restimulated with MN env-type peptides, interisolate recognition was increased.

Target cells: -U-- BC-envAV3

80-

-A

-+-

*I 60

BC-env

BC-VAC-4P160 BC-VAC-LACZ

-0- BC-RP135

.a

40-wo

*

'.

C.s4 .4

C

Effectors: CTLenvAV3 Rn

1:1

3:1

3415

30:1 10:1 Effector: Target Ratio

100:1

FIG. 6. (a) Lysis of murine targets expressing the envelope glycoprotein gene of HIV-1 IIIB by CTLenvAV3. CTLenvAV3 were incubated in a 5-h 51Cr release assay with BC, BC-envAV3 (BC cells transduced with the N2 IIIBenv AV3 retroviral vector), BC-env, BC cells coated with the RP135 (IIIB-specific V3 loop) peptide, or BC cells infected with vaccinia virus-lacZ or vaccinia virus-gpl60 to generate BC-VAC-LACZ and BC-VAC-GP160, respectively. Spontaneous release values were less than 15%; standard errors of the means of triplicate cultures were less than 8% of the mean. (b) Lysis

of Hu/Dd cells infected with prototypic HIV-1 isolates by murine CTLenvAV3. Hu/Dd cells (106) were infected with prototype strains of cell-free HIV-1 or HIV-2 (105 TCID50 obtained from chronically infected H9 cells). Ten days postinfection, HIV-infected cells were used as targets in a 5-h 51Cr release assay using murine CTLenvAV3. Spontaneous release values were less than 22%; standard errors of the means of triplicate cultures were less than 4% of the mean. (c) Lysis of Hu/Dd cells infected with clinical HIV-1 isolates by murine CTLenvAV3. Hu/Dd cells (106) were infected with three different patient strains of cell-free HIV-1 (105 TCID50 obtained from chronically infected MT2 cells). Ten days postinfection, HIV-1-infected cells were used as targets in a 5-h 51Cr release assay using murine CTLenvAV3. Spontaneous release values were less than 25%; standard errors of the means of triplicate cultures were less than 5% of the mean.

3416

J. VIROL.

CHADA ET AL.

These studies examined only PND-specific CTL and showed that it is possible to enhance lysis of targets expressing divergent PND sequences by priming animals with one peptide and restimulating the effectors with a different peptide. In our studies using the recombinant retrovirus system, the IIIB env-immunized splenocytes were restimulated with cells expressing the entire gpl60 protein; therefore, the resulting CTL population was able to recognize cells expressing multiple CTL epitopes contained within gp160 and was not limited to the PND epitope. In this study, we have demonstrated significant CTLenv cross-reactivity between HIV-1 IIIB- and HIV-1 MN-infected targets. Previous studies using vaccinia virus-IIIB env as an immunogen have shown that CTL directed against HIV-1 IIIB env do not efficiently recognize the V3 loop peptide from HIV-1 MN env (39), suggesting that the crossreactivity observed in this study may be due to CTL epitopes that lie outside the PND region. We have previously shown that CTL directed against HIV-1 IIIB env do lyse cells coated with the V3 loop peptide from MN (43). Data obtained from CTLenvAV3 demonstrate that HIV-1 envspecific CTL determinants exist outside the V3 loop and support the hypothesis that potentially conserved CTL determinants outside the V3 loop contribute to the crossreactive lysis of divergent HIV-1 strains. These results complement recent data indicating that cloned CTL from HIV-1-infected patients can recognize at least five distinct regions of gpl60 from HIV-1 IIIB (9). Lysis of HIV-1 MN-infected targets relative to HIV-1 IIIB-infected targets was greater by CTLenvAV3 than CTLenv. This finding may indicate that the IIIB V3 loop can generate substantial CTL reactivity and that when the V3 loop is removed from HIV-1 env, the resulting CTL population can mediate a broader group-specific response. Therefore, the V3 loop may be considered a decoy for CTL reactivity in that when it is removed as an epitope for CTL induction, the remaining CTL population may reflect effectors directed against more conserved epitopes. Moreover, the V3 loop may mask or influence presentation of other epitopes, thereby reducing overall CTL reactivity. In HIV-infected patients, specific epitopes recognized by CD8+ CTL are presented by distinct HLA molecules (19, 23, 32). This situation clearly does not occur in the Hu/Dd system, in which epitopes responsible for lysis of target cells infected with eight different HIV-1 strains were presented by the murine H-2Dd class I MHC molecule. The epitopic peptide-binding pocket of H-2Dd appears to be able to bind env epitopes from a wide variety of HIV-1 strains and present multiple epitopes that are recognized by CTLenv and CTLenvAV3, whereas different human class I molecules appear to be responsible for presenting distinct HIV-1 env CTL determinants. However, recent data have suggested that HLA-A2 can present more than one epitope from HIV-1

(6, 7, 23). Development of the Hu/Dd cell line has allowed the analysis of CTL cross-reactivity with use of divergent prototypic and clinical HIV-1 isolates. In addition, the Hu/Dd system may provide a means of monitoring the stability of CTL epitopes from HIV-1 strains relative to HIV-1 IIIB. In further studies, we have examined a series of sequential

env

HIV-1 isolates obtained from AIDS patients. Although individual HIV-1 isolates vary with respect to amino acid sequence and drug resistance, Hu/Dd target cells infected with all the sequential isolates are recognized and lysed by CTLenv (4b). We have also found that Hu/Dd target cells infected with African HIV-1 isolates can be recognized by CTLenv (8a). The extent of CTL cross-reactivity observed

in this study raises the possibility that delivery of an HIV-1 IIIB env immunogen to infected individuals may induce or augment a group-specific CTL response that can combat the divergent quasispecies of HIV-1 and provide significant immunotherapeutic benefit. ACKNOWLEDGMENTS

We thank D. Richman, D. Looney, and A. Langlois for HIV-1 strains; B. Moss and S. Chakrabarti for vaccinia virus-gp160; D. Kuritzkes for vaccinia virus-lacZ; R. Axel for the HeLa-T4+ line; R. Dutton and G. Thor for anti-Dd antibodies 7.2.14 and 34-5-8S; S. Hedrick for the H-2Dd gene; T. Matthews for confirming identity of the IIIB and MN strains; J. Norberg for FACStar analysis; Susan Stern for help in acquiring reagents; and M. Bevan, S. Hedrick, and M. Irwin for comments on the manuscript. The prototypic HIV-1 strains were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID. This work is part of a joint project between Viagene Inc. and the Green Cross Corporation (Osaka, Japan). REFERENCES 1. Aldovini, A., and B. D. Walker. (ed.). 1990. Techniques in HIV research. Stockton Press, New York. 2. Baltimore, D., and M. B. Feinberg. 1989. HIV-1 revealed; toward a natural history of the infection. N. Engl. J. Med. 321:1673-1675. 2a.Bates, M. B., S. R. Jennings, Y. Tanaka, M. J. Tevethia, and S. S. Tevethia. 1988. Recognition of SV40 T antigen synthesized during viral lytic cycle in monkey kidney cells expressing mouse H-2K and H-2Dbtransfected genes by SV40-specific cytotoxic T lymphocytes leads to the abrogation of virus life cycle. Virology 162:197-205. 3. Blochlinger, K., and H. Diggleman. 1984. Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eukaryotic cells. Mol. Cell. Biol. 4:29292931. 4. Byrne, J. A., and M. B. Oldstone. 1984. Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus in vivo. J. Virol. 51:682-686. 4a.Chada, S., and C. DeJesus. Unpublished data. 4b.Chada, S., et al. Unpublished data. 5. Clark, S. J., M. Saag, W. D. Decker, S. Campbell-Hill, J. L. Roberson, P. J. Veldkamp, J. C. Kappes, B. H. Hahn, and G. M. Shaw. 1991. Cytopathic virus in plasma of patients with symptomatic primary HIV-1 infection. N. Engl. J. Med. 324:954-960. 6. Clerici, M., D. R. Lucey, R. A. Zajac, R. N. Boswell, H. M. Gebel, H. Takahashi, J. A. Berzofsky, and G. M. Shearer. 1991. Detection of cytotoxic T lymphocytes specific for synthetic peptides of gpl60 in HIV-seropositive individuals. J. Immunol. 146:2214-2219. 7. Dadaglio, G., A. Leroux, P. Langlade-Demoyen, E. M. Bahraoui, F. Traincard, R. Fisher, and F. Plata. 1991. Epitope recognition of conserved HIV envelope sequences by human cytotoxic T lymphocytes. J. Immunol. 147:2302-2309. 8. Dai, L. C., K. West, R. Littaua, K. Takahashi, and F. A. Ennis. 1992. Mutation of human immunodeficiency virus type 1 at amino acid 585 on gp4l results in loss of killing by CD8+ A24-restricted cytotoxic T lymphocytes. J. Virol. 66:3151-3154. 8a.DeJesus, C., S. Chada, and D. Waters. Unpublished data. 9. Earl, P. L., S. Koenig, and B. Moss. 1991. Biological and immunological properties of human immunodeficiency virus type 1 envelope protein: analysis of proteins with truncations and deletions expressed by recombinant vaccinia viruses. J. Virol. 65:31-41. 10. Gallo, R. C., S. Z. Salahaddin, M. Popovic, G. M. Shearer, M. Kaplan, B. Haynes, T. J. Palker, R. Redfield, J. Oleske, B. Safai, G. White, P. Foster, and P. D. Markham. 1984. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224:500503.

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