The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes.

The viral regulatory gene, nef, is unique to the human immunodeficiency viruses (HIV) and their related primate lentiviruses. Expression of the nef gene has been shown to be essential to the maintenance of high levels of virus replication and the development of pathogenesis in the animal model of simian immunodeficiency virus (SIV) infection. In contrast to this in vivo model, the use of standard T cell culture systems to study nef function in vitro has produced a spectrum of contradictory results, and has failed to demonstrate a significant positive influence of nef on viral life cycle. We have developed a cell model to study regulation of HIV-1 replication that we believe reflects more accurately virus-cell interactions as they occur in vivo. Our experimental system used acute virus infection of purified, quiescent CD4 lymphocytes and subsequent induction of viral replication through T cell activation. With this cell model, NL4-3 virus clones with open and mutated nef reading frames were compared for replication competence. The clones with nef mutations showed reproducible and significant reductions in both rates of growth and maximal titers achieved. The degree of reduced replication was dependent on initial virus inoculum and the timing of T cell activation. The influence of nef was highly significant for induction of virus replication from a latent state within resting CD4 cells. Its effect was less apparent for virus infection of fully proliferating CD4 cells. This study demonstrates that nef confers a positive growth advantage to HIV-1 that becomes readily discernable in the primary cell setting of virus induction through T cell activation. The experimental cell model, which we describe here, provides not only a means to study nef function in vitro, but also provides important clues to the function of nef in HIV infection in vivo.

Viral dynamics of acute HIV-1 infection.

Viral dynamics were intensively investigated in eight patients with acute HIV infection to define the earliest rates of change in plasma HIV RNA before and after the start of antiretroviral therapy. We report the first estimates of the basic reproductive number (R(0)), the number of cells infected by the progeny of an infected cell during its lifetime when target cells are not depleted. The mean initial viral doubling time was 10 h, and the peak of viremia occurred 21 d after reported HIV exposure. The spontaneous rate of decline (alpha) was highly variable among individuals. The phase 1 viral decay rate (delta(I) = 0.3/day) in subjects initiating potent antiretroviral therapy during acute HIV infection was similar to estimates from treated subjects with chronic HIV infection. The doubling time in two subjects who discontinued antiretroviral therapy was almost five times slower than during acute infection. The mean basic reproductive number (R(0)) of 19.3 during the logarithmic growth phase of primary HIV infection suggested that a vaccine or postexposure prophylaxis of at least 95% efficacy would be needed to extinguish productive viral infection in the absence of drug resistance or viral latency. These measurements provide a basis for comparison of vaccine and other strategies and support the validity of the simian immunodeficiency virus macaque model of acute HIV infection.

HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function.

The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

Patient with 13 chromosome deletion: evidence that the retinoblastoma gene is a recessive cancer gene.

Although a constitutional chromosomal deletion including 13q14 has been found to date in all retinoblastoma patients whose esterase D activity is 50 percent of normal, one female patient has been found who has 50 percent esterase D activity in all normal cells examined but no deletion of 13q14 at the 550-band level. Therefore, she has the smallest constitutional chromosomal deletion within 13q14 that is associated with susceptibility to retinoblastoma. Two stem lines were identified in a retinoblastoma from this patient, and each one had a missing 13 chromosome. No detectable esterase D activity was found in the tumor, indicating that the normal nondeleted 13 chromosome was lost in both stem lines. Thus the data from this patient not only show that there is a total loss of genetic information at the location of the retinoblastoma gene within the tumor, but also imply that recessive genes may play an important role in the development of certain human tumors including retinoblastoma.

Recovery of replication-competent HIV despite prolonged suppression of plasma viremia.

In evaluating current combination drug regimens for treatment of human immunodeficiency virus (HIV) disease, it is important to determine the existence of viral reservoirs. After depletion of CD8 cells from the peripheral blood mononuclear cells (PBMCs) of both patients and normal donors, activation of patient CD4 lymphocytes with immobilized antibodies to CD3 and CD28 enabled the isolation of virus from PBMCs of six patients despite the suppression of their plasma HIV RNA to fewer than 50 copies per milliliter for up to 2 years. Partial sequencing of HIV pol revealed no new drug resistance mutations or discernible evolution, providing evidence for viral latency rather than drug failure.

Preferential replication of HIV-1 in the CD45RO memory cell subset of primary CD4 lymphocytes in vitro.

The ability of HIV-1 to establish an infection and replicate to high copy number in CD4 lymphocytes is dependent on both the activation state of the cell and virus-encoded regulatory proteins that modulate viral gene expression. To study these required virus-cell interactions, we have used an in vitro cell model of acute HIV infection of quiescent, primary CD4 lymphocytes and subsequent induction of T cell activation and virus replication by lectin or CD3 receptor cross-linking. Experiments were done to determine if the capacity of HIV to establish infection and complete replication was impacted by the maturational state of the CD4 cell target or the specific signal induction pathway engaged during activation. Primary CD4 cells were FACS-sorted into the major phenotypic subsets representative of memory (CD45RO) and naive (CD45RA) cells. Levels of virus replication were compared between infection with wild-type NL4-3 virus and an isogenic mutant containing a deletion in nef regulatory gene. PHA mitogen stimulation was compared with anti-CD3, with and without anti-CD28 costimulation, for induction of cell proliferation and virus replication. In both infected and uninfected cells, the RA cell subset exhibited significantly greater response to CD3/CD28 stimulation than did the RO cell subset. In contrast, the majority of virus replication occurred consistently in the RO cell subset. Deletion of HIV nef function caused a severe reduction in viral replication, especially in the RA naive cell subset after CD3 induction. PCR analysis of viral DNA formation, during infection of quiescent cells, demonstrated that the observed differences in HIV replication capacity between RO and RA cell subsets were not due to inherent differences in cell susceptibility to infection. Our results indicate that HIV replication is enhanced selectively in CD45RO memory phenotype cells through the probable contribution of specialized cellular factors which are produced during CD3-initiated signal transduction.

Suppression of spontaneous in vitro transformation of autologous leukocytes by plasma from convalescent and postconvalescent infectious mononucleosis patients.

All 14 plasma samples from 13 convalescent and postconvalescent infectious mononucleosis (IM) patients suppressed the spontaneous in vitro transformation of autologous leukocytes. In contrast, only 2 of 8 plasma samples from patients with acute IM suppressed this transformation. All 7 patients whose blood was tested both in the acute and convalescent or postconvalescent phases of IM showed either a conversion in transformation suppression status from negative to positive or an increase in the strength of transformation suppression. Thus recovery from IM appeared to be associated with the ability of plasma to suppress the in vitro spontaneous transformation of autologous leukocytes.

Depression of the generation of cell-mediated cytotoxicity by macrophage-like suppressor cells in bladder carcinoma patients.

Depressed T-lymphocyte function as assessed by delayed-type hypersensitivity reactions and in vitro proliferative response to mitogens is a characteristic finding in many types of solid tumors, including bladder carcinoma. Peripheral blood leukocytes from 16 patients with transitional cell carcinoma of the bladder were compared with age-matched, control subjects. Both the unfractionated leukocytes containing 10 to 30% monocytes and the lymphocyte-enriched preparations, obtained by monocyte depletion with iron filing ingestion, were analyzed. Mixed leukocyte culture-induced cytotoxicity was depressed in the patient group; the amount of depression was directly correlated to the extent of the disease. In patients who underwent surgical removal of tumor, the mixed leukocyte culture-induced cytotoxicity appeared normal. This mixed leukocyte culture-generated cytotoxic response was a more sensitive indicator of tumor effect than was the induced proliferative response. Removal of phagocytic or adherent monocytes from the responding cell population caused a significant increase in the generated cytotoxicity, especially in those patients with invasive disease. These suppressive effects could be partially reconstituted by quantitative addition of the separated monocytes back to the responding lymphocyte culture. The depressed lymphocyte-mediated cytotoxicity present in these bladder cancer patients was due, in a major part, to a circulating macrophage-like cell with active suppressor function.

The use of three baseline values in intervention studies: application to evaluation of immune modulation therapies.

In studying the effects of a treatment intervention on immunological parameters, we have found that three baseline values are a practical number to obtain on each patient. Three baseline values 1) increase the chances of detecting a statistically significant effect of the intervention, 2) provide an assessment of the daily variability of the assay and patients, and 3) enable the identification of individual patients who demonstrate significant changes associated with the intervention.

Lymphocyte cytotoxicity in the peritoneal cavity and blood of patients with ovarian cancer.

After an intensive course of combination chemotherapy, 16 patients with minimal residual ovarian cancer that was documented at second-look laparotomy, had an indwelling Tenckhoff catheter placed and underwent multiple peritoneal saline lavages. Lymphocyte-enriched populations from the peritoneal cavity and peripheral blood were obtained by density gradient centrifugation and examined for cell-surface phenotype and a variety of immune functions, including natural killer cytotoxicity and antibody-dependent cell-mediated cytotoxicity. Phenotypic characterization revealed that peritoneal lymphocytes consisted primarily of T cells and cells bearing receptors for the crystallizable fragment of immunoglobulin G (IgG) (crystallizable fragment-receptor), and contained a very low number of B cells. Peritoneal natural killer lymphocyte cytotoxicity and antibody-dependent cell-mediated cytotoxicity were very low in all but two patients. Incubation of peritoneal lymphocytes with Corynebacterium parvum and interferon in vitro did not result in augmented cytotoxicity against susceptible targets. Supernatants from cultured peritoneal cells of all patients markedly inhibited natural cytotoxic activity of normal donor blood lymphocytes. These results suggest that lymphocytes collected from the peritoneal cavity of patients with minimal residual ovarian cancer are deficient in natural and antibody-dependent cytotoxic effector function. This deficiency may influence the host's ability to control the spread and proliferation of tumor cells in the peritoneal cavity.

Enhancement of spontaneous killer cytotoxicity by soluble factor.

Two-stage stimulation of SK activity of normal human peripheral blood lymphocytes by soluble factors was demonstrated. The first stage was initiated by factors present in supernatant derived from normal B-LCL cultures. Only cell lines which could induce SK activity in culture in an MLR-type reaction had the capacity to produce the active factor. Supernatant factor required adherent cells to cause SK augmentation. The interaction of adherent cells plus supernatant factor resulted in the production of a second soluble factor which stimulated an increase in SK activity in responding lymphocyte populations. This second stage involved a different soluble factor which acted directly on the non-SK, Fc-negative lymphocyte population, and within 3 hr. Data obtained using antisera to interferon (IF) indicated that IF is a component of the second soluble factor, and not of the supernatant factor derived from the B-LCL.

In vitro stimulation of lymphocytes of donors seronegative for Epstein-Barr virus by preparations of inactivated Epstein-Barr virus.

Heat-inactivated preparations of Epstein-Barr virus stimulated human lymphocytes as assayed by incorporation of [(3)H]thymidine. The inactivated Epstein-Barr virus stimulated the lymphocytes of all five seropositive donors, 11 of 14 seronegative donors (aged eight to 26 years), and none of 15 neonates. Control antigens prepared from a human lymphoid cell line devoid of the Epstein-Barr virus genome did not stimulate the lymphocytes of seronegative donors. Fetal calf serum at the concentration used for suspension of Epstein-Barr virus did not stimulate or only minimally stimulated the lymphocytes of seronegative donors. The reactivity of the histocompatibility antigens found on human lymphocytes was abolished by procedures used for inactivation of the virus.

Establishment of a stable, inducible form of human immunodeficiency virus type 1 DNA in quiescent CD4 lymphocytes in vitro.

Human immunodeficiency virus type 1 (HIV-1) possesses the ability to establish a complete infection in nondividing host cells. The capacity of HIV-1 to infect nondividing cells probably contributes significantly to its pathology in vivo, as reflected by infection of peripheral T lymphocytes, tissue macrophages, and microglial cells. However, the in vitro demonstration of the establishment of stable HIV-1 infection in quiescent T cells remains controversial. We have developed a primary T-cell model of acute HIV-1 infection of quiescent CD4 lymphocytes that demonstrates the development of a complete, reverse-transcribed form of virus that is stable for over 10 days in culture. To ensure that our primary cell culture was representative of a quiescent population, the CD4 lymphocyte targets were monitored for membrane expression of activation antigens and for shifts in cell cycle from G0/G1 to S/G2 phase. The presence of viral DNA fragments reflecting progressive reverse transcription was determined by PCR analysis. HIV entered primary CD4 cells rapidly, but viral DNA accumulated slowly in the resting cell cultures. DNA species containing regions of full-length reverse transcription were not detected until 3 to 5 days after infection. In parallel with the appearance of complete viral DNA, spliced RNA transcripts, predominantly of the nef species, were detected by reverse transcriptase PCR amplification. When infected CD4 cells were sorted on the basis of cell cycle analysis of DNA content, the accumulation of a complete viral DNA form was found to occur in both the purified G0/G1-phase cell subset and the cell fraction enriched for the minor S-phase subset. In contrast, spliced viral RNA products could be detected only in the enriched S-phase cell fraction. These results demonstrate that HIV-1 can infect and establish a complete, stable form of viral DNA in primary CD4 lymphocytes in vitro but is blocked from transcription in the absence of cell activation. The findings are consistent with in vivo data from HIV-infected individuals that show the existence of viral DNA predominantly as a stable, extrachromosomal form in T cells of the peripheral circulation.

In vitro differentiation of monocytoid THP-1 cells affects their permissiveness for HIV strains: a model system for studying the cellular basis of HIV differential tropism.

The prototypic macrophage-tropic HIV-1 isolate, HIV-1BaL, cannot replicate in the monocytoid cell line THP-1. After induction of differentiation by a phorbol diester, a fraction of THP-1 cells became permissive to HIV-1BaL. In contrast, this treatment decreased permissiveness for the lymphotropic isolate HIV-1LAI. Viral DNA was not synthesized in unstimulated THP-1 cells, as determined with PCR, suggesting that the block to HIV-1BaL replication in these cells occurred at an early step of the virus replicative cycle prior to or at the level of reverse transcription. Virus binding studies showed that differences in cell permissiveness for HIV-1BaL were not due to altered virus binding. Substantial amounts of HIV-1BaL bound to both undifferentiated and differentiated THP-1 cells, and this binding could not be prevented by blocking with the anti-CD4 antibody Leu3a, which did prevent the binding of HIV-1LAI to CEM T lymphoid cells. While Leu3a was very effective at preventing the infection by HIV-1LAI in CEM cells, it was less effective in preventing HIV-1BaL infection of differentiated THP-1 cells or primary macrophages. Although it is likely that molecules other than CD4 on monocytic cells can mediate binding of macrophage-tropic HIV, the binding of HIV-1BaL to THP-1 cells was not sufficient for infection, because binding was the same in nonpermissive undifferentiated cells as in permissive differentiated cells. Thus, the restriction of viral replication in this model cell system occurs at some step after virion binding. Comparison of differentiated THP-1 cells with their undifferentiated counterparts may provide an approach to defining cellular determinants of HIV host range other than CD4 expression and to characterizing the incompletely defined steps of viral entry.

Suppression of natural killer cell cytotoxicity in the peripheral blood of patients receiving interferon therapy.

Lymphoblast interferon (IFN-alpha) was administered to patients with advanced stages of cancer in a phase I drug toxicity trial. IFN-alpha was given i.m. twice daily at 12-h intervals over a 7-day course of therapy in dosages ranging from 1.5 to 100 X 10(6) U/day. A total of 28 patients was studied, including 9 with breast carcinoma, 11 with other solid tumors, and 8 with lymphoid malignancies. Immune cell parameters were determined for each patient before, during, and up to 20 days after therapy. Leukopenia was evident after 1-2 days of IFN-alpha administration, became maximal after 6-7 days of therapy, and then returned to baseline values by day 13 post-therapy. Circulating natural killer (NK) cell activity was found to increase significantly by 2 h after the initial IFN injection, especially in patients receiving the higher dosages. However, most subjects demonstrated a return to baseline NK levels at 24 h despite the continued presence of elevated serum concentrations of IFN. By day 7 of therapy, NK-cell function was markedly depressed. Following cessation of IFN, NK levels rapidly returned to pretherapy baseline values. Changes in K-cell cytotoxic function (ADCC) tended to parallel those of NK-cell function. Although NK/K-cell function was affected by IFN therapy, no change in the percentage of circulating Fc receptor-bearing cells was found. This indicates that the cytotoxic cells were probably present in the circulation, but were not able to express their lytic function.

Altered cellular immune functions in patients with Down's syndrome.

Cell-associated immunocompetence was evaluated in 42 patients with Down's syndrome (DS) and compared with that of institutionalized patient control and normal control groups. B lymphocytes, as both percent of total lymphoid cells and absolute number, were markedly reduced in patients with DS. T-lymphocyte proliferative response to phytohemagglutinin mitogen also was significantly decreased. This altered response of the lymphocyte population was evident in both the presence and absence of accessory helper monocytes. Increased Fc receptor (FcR) cell activity, as measured by antibody-dependent cellular cytotoxicity was seen in both the patients with DS and the control patients. This probably resulted from common environmental factors such as exposure to infectious factors such as exposure to infectious agents. No change was found in the WBC counts, percentages of lymphocytes and monocytes, percentages of t cells and FcR cells, or in the natural killer cell activity.

Producer-cell modification of human immunodeficiency virus type 1: Nef is a virion protein.

Type 1 human immunodeficiency viruses encoding mutated nef reading frames are 10- to 30-fold less infectious than are isogenic viruses in which the nef gene is intact. This defect in infectivity causes nef-negative viruses to grow at an attenuated rate in vitro. To investigate the mechanism of Nef-mediated enhancement of viral growth rate and infectivity, a complementation analysis of nef mutant viruses was performed. To provide Nef in trans upon viral infection, a CEM derivative cell line (designated CLN) that expresses Nef under the control of the viral long terminal repeat was constructed. When nef-negative virus was grown in CLN cells, its growth rate was restored to wild-type levels. However, the output of nef-negative virus during the first 72 h after infection of CLN cells was not restored, suggesting that provision of Nef within the newly infected cell does not enhance the productivity of a nef-negative provirus. The genetically nef-negative virions produced by the CLN cells, however, were restored to wild-type levels of infectivity as measured in a syncytium formation assay in which CD4-expressing HeLa cells were targets. These trans-complemented, genetically nef-negative virions yielded wild-type levels of viral output following a single cycle of replication in primary CD4 T cells as well as in parental CEM cells. To define the determinants for producer cell modification of virions by Nef, the role of myristoylation was investigated. Virus that encodes a myristoylation-negative nef was as impaired in infectivity as was virus encoding a deleted nef gene. Because myristoylation is required for both membrane association of Nef and optimal viral infectivity, the possibility that Nef protein is included in the virion was investigated. Wild-type virions were purified by filtration and exclusion chromatography. A Western blot (immunoblot) of the eluate fractions revealed a correlation between peak Nef signal and peak levels of p24 antigen. Although virion-associated Nef was detected in part as the 27-kDa full-length protein, the majority of immunoreactive protein was detected as a 20-kDa isoform. nef-negative virus lacked both 27- and 20-kDa immunoreactive species. Production of wild-type virions in the presence of a specific inhibitor of the human immunodeficiency virus type 1 protease resulted in virions which contained only 27-kDa full-length Nef protein. These data indicate that Nef is a virion protein which is processed by the viral protease into a 20-kDa isoform within the virion particle.

The growth advantage conferred by HIV-1 nef is determined at the level of viral DNA formation and is independent of CD4 downregulation.

Recent data on the phenotype of nef-defective HIV-1 in vitro indicate a new function of the Nef gene product: enhancement of viral infectivity. Single-cycle replication studies have suggested that Nef enhances the efficiency of an early step during viral replication, a step that leads to the establishment of viral DNA. To test this interpretation, the accumulation of low-molecular-weight (unintegrated) viral DNA was measured in cells following exposure to wild-type and nef-defective viruses. nef-defective virus accumulated less DNA than the wild type. This difference was observed after as little as 5 hr of exposure to virus. However, the reverse transcriptase activities of wild-type and nef-defective viruses were equal when measured in cell-free assays using either exogenous or endogenous templates. In addition, the abilities of these viruses to bind and enter cells were not significantly different. Together, these data suggest that Nef optimizes postentry events that are required for efficient synthesis of viral DNA. To determine if these effects were related to the property of Nef-mediated downregulation of CD4, growth curves of these viruses were determined using cells that express a CD4 molecule unable to respond to Nef. nef-defective virus remained attenuated in these cells, indicating that Nef-mediated downregulation of CD4 is not required for Nef-mediated enhancement of viral propagation in vitro.

Alterations in cytotoxic and phenotypic subsets of natural killer cells in acquired immune deficiency syndrome (AIDS).

Testing of cytotoxic function using a panel of natural killer (NK)-sensitive target cells, including a unique herpes simplex virus-infected Raji-cell target, was performed in conjunction with phenotypic cell analysis by dual-color flow cytometry to characterize the NK system. Subjects included in the study were at risk for or infected with the etiologic agent of the acquired immune deficiency syndrome (AIDS), human immunodeficiency virus (HIV). A generalized defect in NK function was temporally correlated with disease manifestations, as evidenced by deficient NK lytic function in patients with AIDS and AIDS-related complex (ARC). Healthy at-risk subjects, including those seropositive for HIV, exhibited robust NK-cell function. Phenotypic analysis revealed that normal proportions of the NK-associated CD16+ (Leu11) Leu7- and CD16+(Leu11)Leu7+ lymphocyte subsets were maintained throughout the clinical progression of HIV infection. However, the proportion and numbers of cells of the CD8+(Leu2)Leu7+ subset were increased in AIDS, ARC, and healthy at-risk subjects, including those seronegative for HIV. These results are consistent with a qualitative defect in the NK system in AIDS, perhaps secondary to CD4-cell depletion and a concomitant lack of essential accessory factors. The elevation in CD8+(Leu2)/Leu7+ cells is not solely the result of HIV infection and may be a general response to viruses and/or other antigenic stimulation.