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A great deal of interest in the study of human immunodeficiency virus (HIV) pathogenesis has been generated in recent years by the discoveries of chemokines and HIV type 1 (HIV-1) coreceptors (reviewed in reference 1). Identification of CCR5, the coreceptor for macrophage-tropic or non-syncytium-inducing (NSI) strains of HIV-1 came from the initial studies of two HIV-1-exposed but uninfected (EU) subjects whose CD4⁺ lymphocytes remained resistant to NSI isolates of HIV-1 (21). Further studies with these cells have shown that high levels of RANTES, macrophage inflammatory protein 1 (MIP-1), and MIP-1, the ligands for CCR5, and a mutant CCR5 gene expression were together responsible for resistance against NSI viruses (7, 15). Fusin, or CXCR4, the coreceptor for T-cell-tropic or syncytium-inducing (SI) strains of HIV-1, was identified soon after the discovery of CCR5 (8). Stromal-derived factor SDF-1 was reported as the ligand for CXCR4 and was shown to be able to block infections with SI viruses in vitro (2). Studies on large cohorts have demonstrated that a homozygous deletion of the CCR5 gene is protective against HIV-1 disease progression primarily through resistance to NSI viruses (31). Conflicting reports showing protection (35) or enhancement (19) of HIV-1 disease progression as a result of SDF-1 mutation have also been published. Although other chemokines/lymphokines such as macrophage-derived chemokine have also been shown to inhibit HIV-1 to some extent, the ligands for CCR5 and CXCR4 are the only factors known to have any real significance in preventing HIV-1 disease progression in vivo (36). However, several studies have indicated evidence of other, as-yet-unknown, cellular factors that may play important roles in suppressing HIV infections (16, 18).

There are conflicting reports as to whether CD4⁺ T cells can be differentially affected in HIV infection. Although some studies have suggested that selective depletion of T cells expressing specific T-cell receptor (TCR)-V sequences occurs in vivo (9, 11), other studies have found no such selective depletion (22, 23). Even though some evidence suggests that the course of HIV-1 infection can depend on genetic loci linked to major histocompatibility complex (HLA in humans) genes (13), no study thus far has shown whether different CD4⁺ cells from an individual patient can respond differently to HIV infection. Cytokines can also play very complex roles in the regulation of HIV replication. We and others have shown that endogenous production of -chemokines by CD4⁺ T cells from asymptomatic HIV-infected subjects can suppress HIV replication (14, 25). However, -chemokines act only at the virus entry level by blocking the binding of viruses to CCR5 and cannot inhibit virus replication once the virus enters the cell. Furthermore, -chemokines are not effective against SI viruses (1).

A major barrier to studies of the functions of human T cells in HIV-infected patients has been the limited growth capacity of these cells in vitro. In recent years, herpesvirus saimiri (HVS) has been used as a powerful tool to immortalize and study human T cells (reviewed in reference 17). We have shown that CD4⁺ and CD8⁺ T cells from HIV-1-positive patients whose infection is nonprogressive and AIDS patients can also be immortalized by HVS for long-term study (27, 28). We report here studies of multiple CD4⁺ T-cell clones developed from a single HIV-1-positive patient whose infection is nonprogressive. Although phenotypically similar, the response of these CD4⁺ clones is drastically different when both are infected in vitro with SI viruses. While some clones were highly susceptible to SI viruses, other clones were resistant to infection. We also provide evidence that the resistance to SI viruses in the selected CD4⁺ clones occurred at a stage after virus entry. Finally, we show that the resistance against SI viruses is mediated, at least partially, by soluble factors produced by the resistant cells that may not include the ligand SDF-1 or other known cytokines.

Generation of CD4⁺ T-cell clones from a patient whose HIV-1 infection is nonprogressive. Descriptions of HIV-infected patients and the development of T-cell clones have been previously reported (27, 28). The CD4⁺ clones studied here were developed from a single patient with nonprogressing HIV-1 infection (NP1) who has remained HIV-1 positive since 1983, displaying no symptoms and maintaining a stable CD4⁺ T-cell count of greater than 1,000 per µl, without any antiretroviral therapy (28). At the time these clones were generated, NP1's viral load was not detectable. Development of these clones has been described previously (27, 28). Briefly, peripheral blood lymphocytes were isolated from heparinized blood by using a Ficoll-Hypaque gradient (Sigma Chemical, St. Louis, Mo.). Lymphocytes were separated with a plastic adherent for 2 h and were resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml) (all from Life Technologies, Grand Island, N.Y.), and human interleukin-2 (IL-2) (20 units/ml) (Boehringer Mannheim, Indianapolis, Ind.). RO 31-8959, a HIV-1 protease inhibitor (a generous gift of I. Duncan), was also added to the medium (final concentration, 10⁵ mM) to inhibit HIV-1 spread during immortalization (24). Cells were immediately infected with HVS group C strain 488-77 (a gift from R. C. Desrosiers, Harvard Medical School) at a multiplicity of infection of 0.1. At 3 to 5 days after infection, HVS-infected cells were cloned by seeding at 0.5 to 1 cell/well containing 10⁵ X-irradiated allogeneic peripheral blood lymphocytes from normal donors in 96-well plates containing 200 µl of the above medium. The protease inhibitor was maintained in the medium for the initial 3 weeks of the cloning process. The growing clones were expanded without any further stimulation with antigen/mitogen. MHCD4, an HVS-immortalized CD4⁺ clone from an HIV-negative donor (26, 30), and the CD4⁺ clones derived from an AIDS patient (27) examined in this report have also been previously described.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Expression of CD4 in clones from NP1 or MHCD4 cells. Cells were stained with fluorescein isothiocyanate-labeled anti-CD4 antibodies or isotype control antibodies (data not shown). 1, NP1-2; 2, NP1-3; 3, NP1-4; 4, NP1-5; 5, NP1-6; 6, MHCD4.

Variable resistance against SI viruses. We next tested the SI virus infectibility of the CD4⁺ clones from NP1. Both laboratory and primary isolates of SI viruses were used in this study for infection of CD4⁺ clones. The laboratory SI strain human T-cell leukemia virus IIIB (IIIB) (donated by R. C. Gallo, University of Maryland) was propagated in H9 cells. Another laboratory SI strain, NL4/3, has been described previously (4). P13, an SI clinical isolate used in this study, has been described previously (33). CD4⁺ clones were infected with SI viruses at 0.5 pg/cell for 2 h at 37°C, washed two times, and resuspended at 3 × 10⁵ cells/ml in 12-well plates. An HVS-immortalized CD4⁺ clone (MHCD4) from an uninfected donor which is susceptible to IIIB virus infection was also used as a control (26). Viable cells were counted by trypan blue exclusion, and supernatants were collected every 3 to 4 days and stored at 80°C. HIV-1 production was measured by p24 ELISA by using a commercial HIV antigen kit (Coulter, Hialeah, Fla.).

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Infection of NP1 clones by SI viruses. Samples of cells (3 × 105/ml) were infected with P13 or IIIB strains of HIV-1 as described in the text. Culture supernatants were collected at regular intervals and assayed for p24 production. (A) Infection with P13 viruses; (B) infection with IIIB viruses.

HIV-resistance is not at virus entry level. Resistance against NSI viruses in CD4⁺ cells from EU subjects was found to be at the virus entry level due to mutant CCR5 coreceptor expression (15). We wondered whether a similar mechanism may be involved in the resistance to SI viruses in the NP1 clones, although it must be emphasized that a global defect in the coreceptor is very unlikely in this case, since some of the clones from the same donor were susceptible to infection. We compared the expression of CXCR4 mRNA in resistant as well as susceptible clones from NP1. Expression of CXCR4 and SDF-1 were tested by reverse transcriptase PCR (RT-PCR) as well as with a fluorescence-activated cell sorter (FACS) using antibodies 12G5 obtained through the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Cellular mRNA was prepared from an equal number (5 × 10⁶) of cells from each clone by using biotin-labeled oligodeoxyribosylthymine-coated magnetic beads from an mRNA isolation kit (Boehringer Mannheim) according to the manufacturer's instructions and was treated with 10 U of RNase-free DNase (Boehringer Mannheim). Random, hexamer-primed cDNA was prepared from 50 µg of mRNA from each sample with an RT-PCR kit (Perkin-Elmer Cetus, Norwalk, Conn.), and cDNA was amplified by 37 cycles of PCR at 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min followed by an additional cycle at 72°C for 10 min for completion of polymerization in a 50-µl volume of primers specific for CXCR4, SDF-1, and -actin (as internal control). The primers for SDF-1 were generated from the human SDF-1 sequence (accession no. U16752), and primers for CXCR4 have been previously described (15). The upstream and downstream primers (synthesized by Life Technologies) were as follows: CXCR4, 5'-GGCTAAAGCTTGGCCTGAGTGCTCCAGTAGCC and 5'-CGTCCTCGAGCATCTGTGTTAGCTGGAGTG, which yield a 1,112-bp fragment, and SDF-1, 5'-CGCCAAGGTCGTGGTCGTGC and 5'-GCCCTTCAGATTGTAGCCCGGC, which yield a 182-bp fragment. The -actin primers have been described previously and yield a 838-bp fragment (28). All samples were run with and without RT to rule out the possibility of any DNA contamination. MHCD4 and SupT1 cells were also used as controls. Amplified products were separated on a 1.2% agarose gel in the presence of 0.5 µg of ethidium bromide per ml and were photographed. In some experiments, mRNA was collected 2 to 3 days after infection with HIV to test whether SDF-1 may be induced after infection. No significant difference in the levels of CXCR4 mRNA expression was observed among susceptible (e.g., NP1-6) or resistant (e.g., NP1-3) clones from NP1 (Fig. 3). HVS-immortalized and SI-susceptible CD4⁺ clones from the uninfected donor (MHCD4) or from the AIDS patient (AD1-13 and AD1-22) (27) also expressed comparable levels of CXCR4 mRNA (Fig. 3). Indeed, using anti-CXCR4 antibody, we did not find any difference in the expression of CXCR4 in several resistant or susceptible CD4⁺ clones by FACS analysis, and all of these clones expressed high and comparable levels of CXCR4 (unpublished observation). These results demonstrate that coreceptor CXCR4 may not contribute to the difference in infection observed in various NP1 clones. We further tested whether the resistant and susceptible clones from NP1 produced different levels of SDF-1. As shown in Fig. 3, both resistant (NP1-6) and susceptible (NP1-3) clones expressed equivalent levels of SDF-1 mRNA, as did the MHCD4 clone from the uninfected donor and SI-virus-susceptible clones AD1-13 and AD1-22 from the AIDS patient. Indeed, no significant change in SDF-1 production was observed in these or other clones from NP1, even after infection with SI viruses (data not shown). Thus, the resistance to SI viruses in the clones from NP1 may not be mediated at the virus entry level through either the inadequate expression of the coreceptor CXCR4 or by the overproduction of SDF-1.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Expression of CXCR4 and SDF-1 in CD4+ clones from a patient with a nonprogressive HIV infection, an AIDS patient, and an uninfected donor. RT-PCR was performed as described in the text with primers specific for CXCR4 (lanes 2 to 6), SDF-1 (lanes 7 to 11), and -actin (lanes 12 to 16). Lanes contain the following: clones NP1-6 (lanes 2, 7, and 12) and NP1-3 (lanes 3, 8, and 13) from the patient with the nonprogressive infection; clones AD1-13 (lanes 4, 9, and 14) and AD1-22 (lanes 5, 10, and 15) from the AIDS patient; and clone MHCD4 (lanes 6, 11, and 16) from the uninfected donor. No DNA contamination was observed when each sample was run without reverse transcriptase (data not shown).

View larger version (131K): [in this window] [in a new window]   FIG. 4.   HIV-1 env-mediated membrane fusion of resistant NP1-2 clones. NP1-2 cells were cocultured with TF228.1.16 cells expressing env from IIIB viruses (A) or with BJAB cells as a control (B). Fusion was indicated by the presence of syncytia in panel A.

View larger version (35K): [in this window] [in a new window]   FIG. 5.   Presence of HIV-1 DNA in NP1-2 clone after infection with IIIB viruses. NP1-2 cells were infected with NL4/3 viruses or kept uninfected as a control. Cellular DNA was isolated after 12 h for PCR amplification with HIV-specific primers for gag (lanes 2 to 4), env (lanes 5 to 7), and -actin as a control (lanes 8 to 10). Lane 1, molecular weight markers; lanes 2, 5, and 7, uninfected NP1-2; lanes 3, 6, and 9, NP1-2 infected with NL4/3 viruses; lanes 4, 7, and 10, positive controls from chronically infected H9-IIIB cells. HVS-immortalized and HIV-1 CD4+ T cells were used as negative controls for these experiments (data not shown).

HIV-suppression factors. CD4⁺ T cells from asymptomatic HIV-infected subjects can protect themselves against NSI viruses by the overproduction of -chemokines (14, 25). As discussed above, we did not find overexpression of SDF-1, the ligand for CXCR4, in any of the resistant clones (Fig. 3). To test whether the HIV-resistant CD4⁺ clones were producing any diffusible anti-HIV factors, we made use of a transwell system as previously described (30). In these experiments, cocultures were performed in two-chambered wells separated by a 0.45-µm-pore-size insert (Millipore, Bedford, Mass.), so that cells in the upper and lower chambers could not come into direct contact but any soluble factor(s) produced by cells on one side could diffuse and act upon cells on the other side. To rule out HVS-induced factors, HVS-transformed MHCD4 cells, which are highly susceptible to infection with IIIB and NL4/3 viruses and produce high levels of HIV-1, were used as the target (26). MHCD4 cells were infected with NL4/3 viruses, washed, and put in the bottom chamber (10⁵ cells/well), while an equal number of cells from different CD4⁺ clones from NP1 were put in the upper chamber. Control chambers contained only medium. Supernatants were collected at various time points from the bottom chamber and assayed for p24 production as described above. As summarized in Table 2, HIV production by MHCD4 cells in the bottom chamber was strongly inhibited when either of the resistant clones, NP1-2 or NP1-3, was placed in the upper chamber. Virus production was also inhibited, albeit at a lower level, when the other resistant clone, NP1-5, was put in the upper chamber. In contrast, when either of the two susceptible clones, NP1-4 or NP1-6, was put in the upper chamber, no inhibition of virus production was observed. Indeed, the level of HIV-1 production was enhanced when clone NP1-4 or NP1-6 was put in the upper chamber, probably as a result of the reinfection of these clones. Taken together, these results suggest that soluble factors produced by resistant NP1 clones potently suppress SI viruses at a stage after virus integration. However, whether these antiviral factors are produced constitutively or only after stimulation with HIV-1 remains unclear, as when we directly added supernatants from the resistant clones to NL4/3-infected cells, no significant inhibition of virus production was observed (data not shown), indicating that the production of these suppression factors may be triggered by HIV antigens. However, it is also possible that the HIV suppression factors that are produced constitutively by the resistant clones have short half-lives and that clones need a continuous supply for effective HIV inhibition.