Contributions of Nonhuman Primates to Research on Aging.

Aging is the biological process of declining physiologic function associated with increasing mortality rate during advancing age. Humans and higher nonhuman primates exhibit unusually longer average life spans as compared with mammals of similar body mass. Furthermore, the population of humans worldwide is growing older as a result of improvements in public health, social services, and health care systems. Comparative studies among a wide range of organisms that include nonhuman primates contribute greatly to our understanding about the basic mechanisms of aging. Based on their genetic and physiologic relatedness to humans, nonhuman primates are especially important for better understanding processes of aging unique to primates, as well as for testing intervention strategies to improve healthy aging and to treat diseases and disabilities in older people. Rhesus and cynomolgus macaques are the predominant monkeys used in studies on aging, but research with lower nonhuman primate species is increasing. One of the priority topics of research about aging in nonhuman primates involves neurologic changes associated with cognitive decline and neurodegenerative diseases. Additional areas of research include osteoporosis, reproductive decline, caloric restriction, and their mimetics, as well as immune senescence and chronic inflammation that affect vaccine efficacy and resistance to infections and cancer. The purpose of this review is to highlight the findings from nonhuman primate research that contribute to our understanding about aging and health span in humans.

Analysis of Gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex.

A tetrameric recombinant major histocompatibility complex (MHC) class I-peptide complex was used as a staining reagent in flow cytometric analyses to quantitate and define the phenotype of Gag-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of simian immunodeficiency virus macaque (SIVmac)-infected rhesus monkeys. The heavy chain of the rhesus monkey MHC class I molecule Mamu-A*01 and beta2-microglobulin were refolded in the presence of an SIVmac Gag synthetic peptide (p11C, C-M) representing the optimal nine-amino acid peptide of Mamu-A*01-restricted predominant CTL epitope to create a tetrameric Mamu-A*01/p11C, C-M complex. Tetrameric Mamu-A*01/p11C, C-M complex bound to T cells of SIVmac-infected, Mamu-A*01(+), but not uninfected, Mamu-A*01(+), or infected, Mamu-A*01(-) rhesus monkeys. Specific staining of peripheral blood mononuclear cells (PBMC) from SIVmac-infected, Mamu-A*01(+) rhesus monkeys was only found in the cluster of differentiation (CD)8alpha/beta+ T lymphocyte subset and the percentage of CD8alpha/beta+ T cells in the peripheral blood of four SIVmac-infected, Mamu-A*01+ rhesus monkeys staining with this complex ranged from 0.7 to 10.3%. Importantly, functional SIVmac Gag p11C-specific CTL activity was seen in sorted and expanded tetrameric Mamu-A*01/p11C, C-M complex-binding, but not nonbinding, CD8alpha/beta+ T cells. Furthermore, the percentage of CD8alpha/beta+ T cells binding this tetrameric Mamu-A*01/p11C, C-M complex correlated well with p11C-specific cytotoxic activity as measured in both bulk and limiting dilution effector frequency assays. Finally, phenotypic characterization of the cells binding this tetrameric complex indicated that this lymphocyte population is heterogeneous. These studies indicate the power of this approach for examining virus-specific CTLs in in vivo settings.

Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes.

Clinical evidence suggests that cellular immunity is involved in controlling human immunodeficiency virus-1 (HIV-1) replication. An animal model of acquired immune deficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)-infected rhesus monkey, was used to show that virus replication is not controlled in monkeys depleted of CD8+ lymphocytes during primary SIV infection. Eliminating CD8+ lymphocytes from monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincident with the reappearance of SIV-specific CD8+ T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of vaccination approaches for preventing infection that will elicit these immune responses.

Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination.

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in containing human immunodeficiency virus-1 (HIV-1) replication in infected individuals, strategies are being pursued to elicit virus-specific CTLs with prototype HIV-1 vaccines. Here, we report the protective efficacy of vaccine-elicited immune responses against a pathogenic SHIV-89.6P challenge in rhesus monkeys. Immune responses were elicited by DNA vaccines expressing SIVmac239 Gag and HIV-1 89.6P Env, augmented by the administration of the purified fusion protein IL-2/Ig, consisting of interleukin-2 (IL-2) and the Fc portion of immunoglobulin G (IgG), or a plasmid encoding IL-2/Ig. After SHIV-89.6P infection, sham-vaccinated monkeys developed weak CTL responses, rapid loss of CD4+ T cells, no virus-specific CD4+ T cell responses, high setpoint viral loads, significant clinical disease progression, and death in half of the animals by day 140 after challenge. In contrast, all monkeys that received the DNA vaccines augmented with IL-2/Ig were infected, but demonstrated potent secondary CTL responses, stable CD4+ T cell counts, preserved virus-specific CD4+ T cell responses, low to undetectable setpoint viral loads, and no evidence of clinical disease or mortality by day 140 after challenge.

Equivalent induction of telomerase-specific cytotoxic T lymphocytes from tumor-bearing patients and healthy individuals.

Although high frequencies of T lymphocytes specific for certain tumor-associated antigens have been detected in some cancer patients, increasing evidence suggests that these T cells may be functionally defective in vivo and fail to induce meaningful clinical responses. One strategy to overcome this limitation is to target novel antigens that are ignored during the natural antitumor immune response but are nevertheless capable of triggering effector T-cell responses against tumors after optimal presentation by antigen-presenting cells. Here, we show that the telomerase catalytic subunit (hTERT)-a nearly universal tumor antigen identified by epitope deduction rather than from patient immune responses-is immunologically ignored by patients despite progressive tumor burden. Nevertheless, HLA-A2-restricted CTLs against hTERT are equivalently induced ex vivo from patients and healthy individuals and efficiently kill human tumor cell lines and primary tumors. Thus, telomerase-specific T cells from cancer patients are spared functional inactivation because of immunological ignorance. These findings support clinical efforts to target the hTERT as a tumor antigen with broad therapeutic potential.

Dual phasic suppression of viral replication following de novo human immunodeficiency virus type 1 (HIV-1) infection in lymphocytes of asymptomatic HIV-1 carriers.

Replication of human immunodeficiency virus type 1 (HIV-1) is suppressed in asymptomatic HIV-1 carriers (ACs). By using an in vitro experimental system, the mechanism of this suppression was investigated. Following in vitro infection of a laboratory HIV-1 strain, the peripheral blood mononuclear cells (PBMC) of ACs transiently supported a low level of viral replication, then the virus production rapidly decreased. PCR analysis revealed that HIV-1 proviral DNA integrated in the PBMC of ACs following infection gradually decreased. Such tapering consequences of in vitro HIV-1 infection in the PBMC of ACs were abrogated by depletion of CD8+ T cells from the culture. Furthermore, the viruses subsequently produced by the PBMC of an AC were less able to replicate than the virus produced by CD8+ cell-depleted PBMC of the same donor. These observations suggested that the CD8+ T cell-mediated suppression of HIV-1 replication in ACs may involve both cytocidal and cytostatic mechanisms: the former kills the cells producing viruses, and the latter inhibits viral spread by reducing viral infectivity.

Retention of HIV-1 inside infected MOLT-4 cells in association with adhesion-induced cytoskeleton reorganization.

OBJECTIVE: To study the mechanism of the suppression of HIV release during cell-to-cell adhesion. DESIGN AND METHODS: To investigate the effects of cell-to-cell adhesion on HIV release in association with cytoskeletal elements, chronically HIV-infected T cells were cocultured with different adherent cell lines, cultured on a fibronectin-coated surface, or treated with cytochalasin D. The amount of viral protein released in the culture supernatant and retained inside the cells was monitored by a p24 enzyme-linked immunosorbent assay and Western blotting. For F-actin staining, cells were stained with FITC-labelled phalloidine and examined by immunofluorescence microscopy. RESULTS: Cocultivation resulted in a reduced amount of virus in the culture supernatant and induced the retention of viral protein inside the infected cells. On adhesion to cells, the F-actin of the infected cells was polarized towards the cell periphery from a diffuse cytoplasmic distribution. Similar data were obtained when the infected cells were treated with cytochalasin D or adhered to fibronectin. CONCLUSION: Cell-to-cell adhesion induced polarization of F-actin, which might facilitate HIV retention inside infected T cells.

Detection of viral RNA in CD4(-)CD8(-) and CD4(-)CD8(+) lymphocytes in vivo in rhesus monkeys infected with simian immunodeficiency virus of macaques.

A definition of the specific cell types that support HIV replication early in the course of infection will be important for understanding AIDS pathogenesis and designing strategies for preventing infection. Observations have indicated that the population of lymphocytes susceptible to productive infection extends beyond activated CD4(+) T cells. To explore this issue, we have employed laser scanning cytometry technology and the techniques of lymphocyte surface immunophenotyping followed by fluorescent in situ hybridization to detect simian immunodeficiency virus of macaques (SIVmac) RNA in phenotypically defined rhesus monkey lymphocytes. The immunophenotype of productively infected cells in either a rhesus monkey T cell line or in PBMCs infected in vitro with SIVmac was remarkably similar to that observed in productively infected PBMCs obtained from monkeys during primary infection. We observed low levels or no detectable expression of CD4 on cells infected in vitro or on PBMCs of infected monkeys. However, a substantial number of SIVmac-infected PBMCs both in cultured lymphocytes and sampled directly from infected monkeys expressed CD8 but not CD4. These observations are consistent with the possibility that the CD4 molecule may be modulated off the surface of CD4(+)CD8(-) or CD4(+)CD8(+) lymphocytes after infection or that infection occurred via a CD4-independent mechanism. Moreover, there was no preferential expression of CD25 on cells positive for SIVmac RNA, which might have been predicted if replication of the virus was occurring selectively in activated lymphocytes. These results broaden the range of lymphocytes that support productive SIVmac infection to include CD4(-)CD8(-) and CD4(-)CD8(+) subsets, and are consistent with virus replication occurring in nonactivated cells.

An IL-2/Ig fusion protein influences CD4+ T lymphocytes in naive and simian immunodeficiency virus-infected Rhesus monkeys.

The T cell-stimulatory cytokine interleukin 2 (IL-2) is being evaluated as a therapeutic in the clinical settings of HIV infection and cancer. However, the clinical utility of IL-2 may be mitigated by its short in vivo half-life, toxic effects, and high production costs. We show here that an IL-2/Ig fusion protein possesses IL-2 immunostimulatory activity in vitro and a long in vivo half-life. IL-2/Ig treatment of healthy rhesus monkeys induced significant increases in CD4(+) T lymphocyte counts and expression of CD25 by these cells. Short courses of IL-2/Ig treatment of simian immunodeficiency virus (SIV)-infected rhesus monkeys in conjunction with antiretroviral drugs resulted in increased CD25 expression on T lymphocytes, and transient increases in CD4(+) T lymphocyte counts. Plasma viremia did not increase in these treated animals. Treatment of healthy or SIV-infected rhesus monkeys with a plasmid encoding the IL-2/Ig protein did not affect CD4(+) T lymphocytes. These results demonstrate that IL-2/Ig has potential utility as an immunostimulatory therapeutic.

The mycoplasma-related inhibitor of HIV-1 reverse transcriptase has a DNase activity and is present in the particle-free supernatants of contaminated cultures.

Drastic inhibition of the human immunodeficiency virus (HIV) reverse transcriptase (RT) by mycoplasma has been noted in many laboratories causing confusion in data interpretation. The mycoplasma-related inhibitor of HIV-1 RT was identified as a soluble protein in the particle-free supernatant of a contaminated culture. Gel filtration studies revealed the molecular mass of this protein to be about 70 kDa. This RT-inhibitor contained a DNase with strong activity on both linear and circular DNAs. Addition of this inhibitor after completion of reverse transcription still reduced the final outcome of the RT assay significantly, implying that the inhibitory mechanism occurred mainly by its DNase activity. Treatment of the culture with an antimycoplasma drug cured the mycoplasma contamination, removed the RT-inhibitor and abolished the DNase activity.

Chimeric immune receptors (CIRs) specific to JC virus for immunotherapy in progressive multifocal leukoencephalopathy (PML).

Progressive multifocal leukoencephalopathy (PML) is a deadly brain disease caused by the polyomavirus JC (JCV). The aim of this study is to develop 'designer T cells' armed with anti-JCV TCR-based chimeric immune receptors (CIRs) by gene modification for PML immunotherapy. Two T cell lines specific to two dominant CTL epitopes derived from JCV VP1 protein (termed p36 and p100) from an HLA-A0201+ PML survivor were generated for TCR cloning. Two distinct dominant TCR alpha chains (Valpha6 and Valpha12) and a unique TCR beta chain (Vbeta5.1) were cloned from the p36-specific cell line, while only one alpha (Valpha8.6) and one beta (Vbeta2) chains were dominant in the p100-specific line. Retroviral constructs encoding CIRs were created with the extracellular domains of TCR alpha and beta chains fused to the transmembrane and cytoplasmic portions of CD3zeta (ValphaCalphaCD3zeta or VbetaCbetaCD3zeta). Cellular expression and screening for binding specific peptide-HLA-A0201 tetramer confirmed the reactivity of the p100 TCRalphabeta and of one of the two pairs of p36 TCRalphabeta (Valpha12 and Vbeta5.1). Functional tests confirmed CIR-expressing T cells secreted cytokines and expressed potent cytotoxicity on contact with A0201+ B-lymphoblastoid line loaded with peptides and/or with HLA-A0201+ cells expressing native JCV VP1 protein. In conclusion, anti-JCV designer T cells were generated.

Coexistence of fusion receptors for human T-cell leukemia virus type-I (HTLV-I) and human immunodeficiency virus type-1 (HIV-1) on MOLT-4 cells.

Human immunodeficiency virus type-1 (HIV-1) and human T-cell leukemia virus type-I (HTLV-I) have a similar tropism for target cell types, especially for CD4+ T cells. In this study, we provide evidence that receptors of these two viruses exist independently on the target cell. We established an HTLV-I-producing CD8+ T cell line (ILT-8M2) with a remarkable cell fusion capacity. When cocultured with MOLT-4 cells, ILT-8M2 cells induced giant syncytia more efficiently than any other tested HTLV-I-producer cell lines. In contrast to other HTLV-I-producers, ILT-8M2 cells were minimally susceptible to cytopathic effects of HIV-1 due to very low expression of CD4, although they were able to be persistently infected by HIV-1. The indicator MOLT-4 cells are known to respond well to HIV-1-induced cell fusion, but they lose this ability if they become persistently infected with HIV-1 because of the reduction of CD4 receptor expression. ILT-8M2 was, however, still capable of inducing syncytia with the MOLT-4 cells persistently infected by HIV-1 (MOLT-4/IIIB). This syncytium formation was dependent on the HTLV-I-envelope, as it was inhibited by HTLV-I-positive human sera or a monoclonal antibody to HTLV-I gp46 but not by monoclonal antibodies to HIV-1 gp120 or CD4. Moreover, ILT-8M2 cells persistently infected by HIV-1 (ILT-8M2/IIIB) induced both HTLV-I- and HIV-1-mediated syncytia with uninfected MOLT-4 cells. These results suggest that HTLV-I induces cell fusion utilizing receptors on the target cells independent of HIV-1-receptors.

Inefficient transmission of HTLV-I to MOLT-4 cells by cell-free virus and cocultivation.

Transmission of human T cell leukemia virus type I (HTLV-I) to a T cell line (MOLT-4#8) was studied using cell-free virus infection or cocultivation with an HTLV-I-transformed T cell line (MT-2). Immunofluorescence and FACS analyses showed that HTLV-I was efficiently adsorbed onto MOLT-4#8 cells. However, after adsorption, no extrachromosomal viral DNA in the cells was detected by the Southern blot method. In contrast, when MT-2 cells were cocultured with MOLT-4#8 cells, generation of extrachromosomal DNA was clearly observed. These data suggest that the cell-free HTLV-I may have difficulties in penetration, uncoating or reverse transcription. After cocultivation, MOLT-4#8 cells chronically infected with HTLV-I were cloned and analyzed. Only four provirus-positive cell lines were obtained. The transmission rate of the virus by cocultivation seemed to be low in our experimental system, although marked cell fusion was observed. Moreover, none of the cloned cell lines which harbored HTLV-I provirus expressed any viral protein. Inefficient integration and expression of the provirus might be hypothesized as compared with human immunodeficiency virus type 1 transmission.

Specific and independent recognition of U3 and U5 att sites by human immunodeficiency virus type 1 integrase in vivo.

The retroviral attachment (att) sites at viral DNA ends are cis-acting regions essential for proviral integration. To investigate the sequence features of att important for human immunodeficiency virus type 1 (HIV-1) integration in vivo, we generated a series of 25 att mutants of HIV-1 by mutagenesis of the U3, U5, or both boundaries of att. Our results indicated that the terminal 11 or 12 bp of viral DNA are sufficient for specific recognition by HIV-1 integrase (IN) and suggested that IN might recognize each att site independently in vivo.

Double intratracheal instillation of keratinocyte growth factor prevents bleomycin-induced lung fibrosis in rats.

Alveolar re-epithelialization is necessary in the repair of damaged alveolar epithelium after lung injury. Keratinocyte growth factor (KGF) has been shown to be a potent proliferation and differentiation factor for rat alveolar type II cells. The present study examined whether KGF would prevent bleomycin-induced lung fibrosis. Adult rats were anaesthetized and recombinant human KGF (rhKGF) (150 micrograms/kg) or saline was injected intratracheally at 48 h before and 24 h after bleomycin (Bleo, 5 mg/kg) instillation. Seven and 14 days after the last administration, rat lungs were processed for lung physiology, immunohistochemistry, and in situ hybridization. Double instillation of KGF prevented the loss of body weight and reduction in total lung capacity (TLC) due to Bleo, and markedly attenuated the protein accumulation and mRNA expression of collagen types I and III and the decreased expression of surfactant protein mRNAs in the fibrotic lesions of Bleo-treated rats. KGF may play an important role in maintaining alveolar epithelium and repairing the damaged epithelium after lung injury.

In vitro modification of human immunodeficiency virus type 1 (HIV-1) infectivity by the U937 cells.

The effect of host cell factors on infectivity of human immunodeficiency virus type 1 (HIV-1) was studied by infecting a monoblastoid cell line (U937) or a T-cell line (MOLT-4) with a highly infective single clone of HIV-1 and comparing the infectivity of the produced viruses to different cell lines. Chronically infected U937 cells consistently produced viruses with minimal infectivity. This phenotypic change was host-dependent as the back-passage of the U937-produced low infective viruses into MOLT-4 cells resulted in regaining their original high infectivity. Southern and Northern blot analyses of the HIV-1 grown in U937 cells did not reveal any genomic difference between it and the virus grown it MOLT-4 cells. The radioimmunoprecipitation analysis of viral proteins showed that the HIV-1-infected U937 cells had a different pattern of envelope glycoproteins and core proteins, which well correlated with the low infectivity of the produced viruses. This experimental system using MOLT-4 and U937 cell lines would be useful to further explore host cell factor(s) which play an important role in the regulation of HIV-1 infectivity.

Exposure of p19 matrix protein of human T-cell leukemia virus type I (HTLV-I) on the surface of MOLT-4#8 cells after virus adsorption.

The p19 matrix (MA) protein of human T-cell leukemia virus type I (HTLV-I) was exposed on the surface of MOLT-4#8 cells in the very early step of the virus infection. Transfer of the virus-binding MOLT-4#8 cells from 4 degrees C to 37 degrees C resulted in increased detection of the viral gp46 and p19 MA protein on the cells, which was, however, inhibited by 4 degrees C or cytochalasin B treatment. These data showed that increased temperature and fluidity of the cell membrane were required for the increased detection of gp46 and p19 after viral adsorption. On the other hand, exposure of the p19 MA protein was not observed on the virus-treated U937 cells although gp46 was detected. This was not due to inefficient binding of the HTLV-I to the U937 cells, since the methanol-fixed cells were p19 MA protein-positive. MOLT-4#8 cells induced marked cell fusion when co-cultured with MT-2 cells, but U937 cells induced no fusion. All of these results indicated that these two cell lines differed in the property of plasma membrane in terms of degradation of HTLV-I envelope after viral adsorption. Uncoating of the HTLV-I might occur on the plasma membrane, especially on MOLT-4#8 cells.

Analysis of 3' terminals of human immunodeficiency virus type 1 transcripts in persistently infected cells.

To examine the 3' terminal processing of human immunodeficiency virus type 1 (HIV-1) transcripts and the effects of phorbol ester (TPA) on this processing, cellular RNAs from persistently infected T cells (MOLT-4) or promonocytes (U937), with or without TPA treatment, were analyzed. To map the 3' terminals of viral transcripts, the RNA samples were examined by RNase-protection assay with an HIV-1 long terminal repeat (LTR) antisense riboprobe. Without TPA treatment, the viral transcripts initiated at the cap site in 5' LTR and polyadenylated at poly(A) site in 3' LTR were dominantly detected in both types of cells. This analysis demonstrated that some occlusion mechanism inactivating the poly(A) site in 5' LTR might exist in these infected cells. After TPA treatment, we found a dramatic shift in the protected patterns of viral transcripts in MOLT-4 cells, while the shift in U937 cells was less dramatic. These results suggested that the primary factor(s) involved in the observed effect of TPA might be cellular. We also demonstrated that the shift in the protected patterns of viral transcripts was associated with increased steady-state levels of viral transcripts. These results indicated that the factors involved in the TPA-induced shift might have some relation to the trans-activation of HIV-1 by similar substances.

Generation of neutralization-resistant HIV-1 in vitro due to amino acid interchanges of third hypervariable env region.

Antigenic mutants of HIV-1 were isolated from three plaque-cloned viruses by the resistance of the virus to neutralizing mAb 0.5 beta against V3 domain of viral gp120, when the viruses were passaged in the presence of the antibody. However, when chronically infected MOLT-4 cells were treated with 0.5 beta mAb, recovered viruses from the 0.5 beta-treated cells showed no antigenic changes. The extent of genomic variation among antigenically distinct isolates was examined by nucleotide sequencing, which revealed a few base substitutions in 0.5 beta-binding site of all mutants isolated. The predicted amino acid replacements within 0.5 beta reacting epitope (V3 domain) causing the altered antigenicity were also identified for each of three isolates. Particularly, in one of the mutants, the most conserved Gly-Pro-Gly-Arg region located at the center of the V3 domain was changed to Gly-Gln-Gly-Arg. The radioimmunoprecipitation and synthetic peptide analyses revealed that this Pro320----Gln substitution reduced the binding affinity with 0.5 beta, although other mutations observed in the other mutants did not affect the binding affinity in radioimmunoprecipitation. We also observed that nucleic acid substitutions in the V3 domain occurred frequently in the absence of 0.5 beta mAb during our in vitro acute infection system using MT-4 cells.