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Aging is characterized by a loss of bone marrow hematopoietic tissue, systemic chronic inflammation, and higher susceptibility to infectious and noninfectious diseases. We previously reported the tightly regulated kinetics and massive daily production of neutrophils during homeostasis in adult rhesus macaques aged 3 to 19 yr (equivalent to approximately 10 to 70 yr of age in humans). In the current study, we observed an earlier release of recently dividing neutrophils from bone marrow and greater in‐group variability of neutrophil kinetics based on in vivo BrdU labeling in a group of older rhesus macaques of 20–26 yr of age. Comparing neutrophil numbers and circulating cytokine levels in rhesus macaques spanning 2 to 26 yr of age, we found a negative correlation between age and blood neutrophil counts and a positive correlation between age and plasma G‐CSF levels. Hierarchic clustering analysis also identified strong associations between G‐CSF with the proinflammatory cytokines, IL‐1β and MIP‐1α. Furthermore, neutrophils from older macaques expressed less myeloperoxidase and comprised higher frequencies of polymorphonuclear myeloid‐derived suppressor cells (PMN‐MDSCs) compared to the young adult macaques. In summary, we observed an earlier release from bone marrow and a reduced production of neutrophils despite the increased levels of plasma G‐CSF, especially in the elderly rhesus macaques. This lower neutrophil production capacity associated with increased production of proinflammatory cytokines as well as an earlier release of less mature neutrophils and PMN‐MDSCs may contribute to the chronic inflammation and greater susceptibility to infectious and noninfectious diseases during aging. Dysregulated neutrophil kinetics, increasing G‐CSF levels, and higher frequency of PMN‐MDSCs accompany chronic inflammation during aging in rhesus macaques.

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The development of chronic inflammation, called inflammaging, contributes to the pathogenesis of age-related diseases. Although it is known that both B and T lymphocyte compartments of the adaptive immune system deteriorate with advancing age, the impact of aging on immune functions of Th17-type CD161-expressing innate immune cells and their role in inflammaging remain incompletely understood. Here, utilizing the nonhuman primate model of rhesus macaques, we report that a dysregulated Th17-type effector function of CD161+immune cells is associated with leaky gut and inflammatory phenotype of aging. Higher plasma levels of inflammatory cytokines IL-6, TNF-α, IL-1β, GM-CSF, IL-12, and Eotaxin correlated with elevated markers of gut permeability including LPS-binding protein (LBP), intestinal fatty acid binding protein (I-FABP), and sCD14 in aging macaques. Further, older macaques displayed significantly lower frequencies of circulating Th17-type immune cells comprised of CD161+T cell subsets, NK cells, and innate lymphoid cells. Corresponding with the increased markers of gut permeability, production of the type-17 cytokines IL-17 and IL-22 was impaired in CD161+T cell subsets and NK cells, along with a skewing towards IFN-γ cytokine production. These findings suggest that reduced frequencies of CD161+immune cells along with a specific loss in Th17-type effector functions contribute to impaired gut barrier integrity and systemic inflammation in aging macaques. Modulating type-17 immune cell functions via cytokine therapy or dietary interventions towards reducing chronic inflammation in inflammaging individuals may have the potential to prevent or delay age-related chronic diseases and improve immune responses in the elderly population.

Rhesus macaques are physiologically similar to humans and, thus, have served as useful animal models of human diseases including cardiovascular disease. The purpose of this study was to characterize the distribution, composition, and phenotype of macrophages in heart tissues of very young (fetus: 0.5 years, n= 6), young adult (2–12 years, n= 12), and older adult (13–24 years, n= 9) rhesus macaques using histopathology and immunofluorescence microscopy. Results demonstrated that macrophages were uniformly distributed throughout the heart in animals of all age groups and were more prevalent than CD3‐positve T‐cells and CD20‐positive B‐cells. Macrophages comprised approximately 2% of heart tissue cells in the younger animals and increased to a mean of nearly 4% in the older adults. CD163‐positive macrophages predominated over HAM56‐positive and CD206‐positive macrophages, and were detected at significantly higher percentage in the animals between 13 and 24 years of age, as well as in heart tissues exhibiting severe histopathology or inflammation in animals of all age groups. In vivo dextran labeling and retention indicated that approximately half of the macrophages were longer lived in healthy adult heart tissues and may comprise the tissue‐resident population of macrophages. These results provide a basis for continued studies to examine the specific functional roles of macrophage subpopulations in heart tissues during homeostasis and in cardiovascular disease for then developing intervention strategies. Macrophages comprise 2–4% of cells in healthy young and adult rhesus macaque heart tissues, are uniformly distributed, and increase in percentage with age.

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Accumulation of IM and increased monocyte turnover during SIV infection is associated with chronic pulmonary inflammation and tissue damage during disease progression. We recently reported that increasing blood monocyte turnover that was associated with tissue macrophage death better predicts terminal disease progression in adult SIV‐infected macaques than does declining CD4+T cell levels. To understand better mechanisms of pathogenesis, this study relates severity of lung‐tissue damage to the ratio, distribution, and inflammatory responses of lung macrophage subsets during SIV infection in rhesus macaques exhibiting varying rates of monocyte turnover. In vivo BrdU incorporation was used to evaluate kinetics of monocyte/tissue macrophage turnover. Tissue damage was scored microscopically from H&E‐stained lung‐tissue sections, and cytokine expression was examined via immunohistochemistry and confocal microscopy. Increased monocyte turnover in SIV‐infected rhesus macaques significantly correlated with severity of lung‐tissue damage, as exhibited by perivasculitis, vasculitis, interstitial pneumonia, alveolar histiocytosis, foamy macrophages, multinucleated giant cells, fibrin, and edema in the alveoli. In addition, the higher monocyte turnover correlated with declining AI ratio, increased accumulation of IM in the perivascular region of the lung, and higher expression of IL‐6 in the IM of the lung tissue exposed to a LPS, calcium ionophore, and tumor promoter combination stimulation ex vivo. Accumulation of IM associated with increasing monocyte turnover during SIV infection appears to contribute to chronic pulmonary inflammation and tissue damage during disease progression to AIDS.

ABSTRACTDespite the availability of annually formulated vaccines, influenza virus infection remains a worldwide public health burden. Therefore, it is important to develop preclinical challenge models that enable the evaluation of vaccine candidates while elucidating mechanisms of protection. Here, we report that naive rhesus macaques challenged with 2009 pandemic H1N1 (pH1N1) influenza virus do not develop observable clinical symptoms of disease but develop a subclinical biphasic fever on days 1 and 5 to 6 postchallenge. Whole blood microarray analysis further revealed that interferon activity was associated with fever. We then tested whether type I interferon activity in the blood is a correlate of vaccine efficacy. The animals immunized with candidate vaccines carrying hemagglutinin (HA) or nucleoprotein (NP) exhibited significantly reduced interferon activity on days 5 to 6 postchallenge. Supported by cellular and serological data, we conclude that blood interferon activity is a prominent marker that provides a convenient metric of influenza virus vaccine efficacy in the subclinical rhesus macaque model.

Data review of the hypothesis that damage in the monocyte/macrophage arm of innate immunity is a key component predicting AIDS progression. Destruction of CD4+T cells is considered to be the main cause of immunodeficiency manifested by opportunistic infections in HIV‐1‐infected humans, as well as in SIV‐infected macaques. We propose that monocyte/macrophage lineage cells also play an important role in the pathogenesis of AIDS, based on our recent work with the SIV/rhesus macaque animal model. We propose that damage to CD4+T cells is important and readily apparent, but damage to monocyte/macrophage lineage cells, although less obvious, may provide the missing link to predict the onset of opportunistic infections and progression to AIDS.

It is widely accepted that destruction of CD4+ T cells and viral load are the primary markers for immunodeficiency in HIV-1–infected humans and in simian immunodeficiency virus (SIV)–infected macaques. However, monocyte/macrophages are also important targets of HIV/SIV infection and a critical link between innate and adaptive immunity. We therefore examined whether changes in cells of the monocyte/macrophage lineage could be linked to the pathogenesis of AIDS in the rhesus macaque model. Here, we show that massive turnover of peripheral monocytes associated with death of tissue macrophages correlates with AIDS progression in macaques. More importantly, the level of monocyte turnover was not linked to the CD4+ T-cell count and was a better predictive marker for AIDS progression than was viral load or lymphocyte activation. Our results show the importance of monocyte/macrophages in the pathogenesis of AIDS and suggest the dynamic changes of the monocyte/macrophages as a new marker for AIDS progression.

It is widely accepted that destruction of CD4+T cells and viral load are the primary markers for immunodeficiency in HIV-1–infected humans and in simian immunodeficiency virus (SIV)–infected macaques. However, monocyte/macrophages are also important targets of HIV/SIV infection and a critical link between innate and adaptive immunity. We therefore examined whether changes in cells of the monocyte/macrophage lineage could be linked to the pathogenesis of AIDS in the rhesus macaque model. Here, we show that massive turnover of peripheral monocytes associated with death of tissue macrophages correlates with AIDS progression in macaques. More importantly, the level of monocyte turnover was not linked to the CD4+T-cell count and was a better predictive marker for AIDS progression than was viral load or lymphocyte activation. Our results show the importance of monocyte/macrophages in the pathogenesis of AIDS and suggest the dynamic changes of the monocyte/macrophages as a new marker for AIDS progression.

Because of the importance of developing HIV vaccine strategies that generate cytotoxic T lymphocyte (CTL) responses with a maximal breadth of epitope recognition, we have explored a variety of novel strategies designed to overcome the usual propensity of CTLs to focus recognition on a limited number of dominant epitopes. In studies of rhesus monkeys expressing the Mamu-A*01 MHC class I allele, we show that variously configured multiepitope plasmid DNA vaccine constructs elicit CTL populations that do not evidence skewing of recognition to dominant epitopes. Nevertheless, repeated boosting of these vaccinated monkeys with different live recombinant vaccine vectors uncovers and amplifies the usual CTL epitope dominance hierarchy. Importantly, in vitro peptide stimulation of peripheral blood mononuclear cells from monkeys that have received only a multiepitope plasmid DNA priming immunization uncovers this dominance hierarchy. Therefore, the dominance hierarchy of the vaccine-elicited epitope-specific CTL populations is inherent in the T lymphocytes of the monkeys after initial exposure to epitope peptides, and the ultimate breadth of epitope recognition cannot be modified thereafter. This finding underscores the enormous challenge associated with increasing the breadth of CTL recognition through vaccination.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the CNS of immunosuppressed individuals caused by the polyomavirus JC (JCV). In previous studies, we showed that JCV-specific cytotoxic T lymphocytes (JCV-specific CTL) were associated with a favourable outcome in patients with PML. However, these CTL had been assessed in PML survivors more than 1 year after the onset of disease and we could not determine whether this immune response was only a surrogate marker for a general recovery of the patient's immune system or a causal factor in the patient's neurological improvement. In this study, we assessed the relationship between JCV-specific CTL detected early in the course of PML and the subsequent course of disease activity. We enrolled 26 patients with possible or proven PML, including 21 HIV+ patients, less than 10 months after the onset of their neurological symptoms (3.7 ± 2.5 months, median ± interquartile range). JCV-specific CTL were detected by either 51Cr release or tetramer staining assay. Patients were then followed prospectively and the clinical course of PML was determined. At the time of their first immune evaluation, we found that 15 patients had detectable JCV-specific CTL. HIV+ patients with JCV-specific CTL had a higher CD4+ T-cell count (215 ± 103/µl) and a lower HIV viral load (144 ± 431 copies/ml) than those without JCV-specific CTL (32 ± 59/µl, P = 0.004 and 43 100 ± 54 778 copies/ml, P = 0.01). Thirteen of these 15 patients with JCV-specific CTL developed clinically quiescent PML, while only two out of 11 without detectable CTL controlled their neurological disease. Therefore, the early detection of JCV-specific CTL had an 87% predictive value for subsequent control of PML, while the absence of such CTL had an 82% predictive value for subsequent active PML (P = 0.0009). Fifteen patients were evaluated less than 4 months after the onset of PML (1.9 ± 1.3 months). Of nine patients with JCV-specific CTL, seven (78%) demonstrated subsequent control of disease, whereas six out of six (100%) without JCV-specific CTL developed progressive PML (P = 0.007). Two to ten CTL assays were performed on PBMC of 11 patients. Of these patients, one had an increase in JCV-specific CTL preceding a significant clinical improvement. In another patient with otherwise stable immune parameters, a decline in JCV-specific CTL preceded an exacerbation of PML. We conclude that JCV-specific CTL can be detected early in PML and can predict control of this disease. Fluctuations of JCV-specific CTL in the blood are associated with variation in disease manifestations. These results indicate that JCV-specific CTL are associated with the control of PML.

We analyzed the prevalence and longitudinal fluctuation of hepatitis B virus (HBV)-specific CD8 T cells in chronic HBV infection using an HLA-A2–HBc18-27 tetramer. Thirty-five HLA-A2-positive patients with chronic HBV infection were divided into 17 HBe antigen (HBeAg)-positive and 18 anti-HBe antibody (anti-HBe)-positive patients. Five HLA-A2-positive normal subjects, five HLA-A2-negative patients with chronic HBV infection, and two HLA-A2-positive patients with acute HBV infection were included as controls. HBc18-27-specific CD8 T cells (c18-27-CD8Ts) were detected at a significantly higher prevalence in patients with anti-HBe (6/18) than in those with HBeAg (1/17), and their frequency reached 0.28% of the total CD8 T cells. The prevalence was significantly higher in patients with HBV DNA below 4.0 log genome equivalents (LGE)/ml (5/12) than in those with HBV DNA above 4.0 LGE/ml (2/23). The frequency of c18-27-CD8Ts was consistently higher in liver-infiltrating lymphocytes, ranging from 0.18 to 1.28%, than in autologous peripheral blood lymphocytes. Longitudinal analysis of patients with acute flare-up demonstrated that the elevation of alanine aminotransferase (ALT) was intimately associated with the expansion of c18-27-CD8Ts. Phenotypic analysis revealed that most c18-27-CD8Ts during acute flare-up expressed HLA-DR and CCR5, while those during low-ALT periods showed low expression. Furthermore, most liver-infiltrating c18-27-CD8Ts were positive for HLA-DR and CCR5, suggesting selective recruitment of activated c18-27-CD8Ts into the liver. In conclusion, HBV-specific CD8 T cells play an important role in the suppression of virus replication, and acute flare-up is associated with the expansion and activation of HBV-specific memory cells.

ABSTRACTSince most human immunodeficiency virus (HIV) infections are initiated following mucosal exposure to the virus, the anatomic containment or abortion of an HIV infection is likely to require vaccine-elicited cellular immune responses in those mucosal sites. Studying vaccine-elicited mucosal immune responses has been problematic because of the difficulties associated with sampling T lymphocytes from those anatomic compartments. In the present study, we demonstrate that mucosal cytotoxic T lymphocytes (CTL) specific for simian immunodeficiency virus (SIV) and simian HIV can be reproducibly sampled from intestinal mucosal tissue of rhesus monkeys obtained under endoscopic guidance. These lymphocytes recognize peptide-major histocompatibility complex class I complexes and express gamma interferon on exposure to peptide antigen. Interestingly, systemic immunization of monkeys with plasmid DNA immunogens followed by live recombinant attenuated poxviruses or adenoviruses with genes deleted elicits high-frequency SIV-specific CTL responses in these mucosal tissues. These studies therefore suggest that systemic delivery of potent HIV immunogens may suffice to elicit substantial mucosal CTL responses.

ABSTRACTAlthough recent evidence has confirmed the importance of cytotoxic T-lymphocyte (CTL) responses in controlling human immunodeficiency virus type 1 and simian immunodeficiency virus replication, the relevance of the epitopic breadth of those CTL responses remains unexplored. In the present study, we sought to determine whether vaccination can expand CTL populations which recognize a repertoire of viral epitopes that is greater than is typically generated in the course of a viral infection. We demonstrate that potent secondary CTL responses to subdominant epitopes are rapidly generated following a pathogenic simian-human immunodeficiency virus challenge of rhesus monkeys vaccinated with plasmid DNA or recombinant modified vaccinia virus Ankara vaccines. These data indicate that prior vaccination can increase the breadth of the CTL response that evolves after an AIDS virus infection.

Although systemic virus-specific cytotoxic T lymphocyte (CTL) responses are of critical importance in controlling virus replication in individuals infected with human immunodeficiency virus 1 (HIV-1), little is known about this immune response in the gastrointestinal (GI) tract. This study investigated the GI tract CTL response in a nonhuman primate model for HIV-1 infection, simian immunodeficiency virus (SIV)–infected rhesus monkeys. Lymphocytes from duodenal pinch biopsy specimens were obtained from 9 chronically SIVmac-infected rhesus monkeys and GI tract lymphocytes were harvested from the jejunum and ileum of 4 euthanized SIVmac-infected rhesus monkeys. Lymphocytes were also assessed in GI mucosal tissues by in situ staining in histologic specimens. SIVmac Gag-specific CTLs were assessed in the monkeys using the tetramer technology. These GI mucosal tissues of chronically SIVmac-infected rhesus monkeys contained levels of CTLs comparable to those found in the peripheral blood and lymph nodes. The present studies suggest that the CD8+CTL response in GI mucosal sites is comparable to that seen systemically in SIVmac-infected rhesus monkeys.

Although systemic virus-specific cytotoxic T lymphocyte (CTL) responses are of critical importance in controlling virus replication in individuals infected with human immunodeficiency virus 1 (HIV-1), little is known about this immune response in the gastrointestinal (GI) tract. This study investigated the GI tract CTL response in a nonhuman primate model for HIV-1 infection, simian immunodeficiency virus (SIV)–infected rhesus monkeys. Lymphocytes from duodenal pinch biopsy specimens were obtained from 9 chronically SIVmac-infected rhesus monkeys and GI tract lymphocytes were harvested from the jejunum and ileum of 4 euthanized SIVmac-infected rhesus monkeys. Lymphocytes were also assessed in GI mucosal tissues by in situ staining in histologic specimens. SIVmac Gag-specific CTLs were assessed in the monkeys using the tetramer technology. These GI mucosal tissues of chronically SIVmac-infected rhesus monkeys contained levels of CTLs comparable to those found in the peripheral blood and lymph nodes. The present studies suggest that the CD8+ CTL response in GI mucosal sites is comparable to that seen systemically in SIVmac-infected rhesus monkeys.

ABSTRACTThe ability to monitor vaccine-elicited CD8+cytotoxic T-lymphocyte (CTL) responses in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys has been limited by our knowledge of viral epitopes predictably presented to those lymphocytes by common rhesus monkey MHC class I alleles. We now define an SIV and SHIV Nef CTL epitope (YTSGPGIRY) that is presented to CD8+T lymphocytes by the common rhesus monkey MHC class I molecule Mamu-A*02. All seven infectedMamu-A*02+monkeys evaluated demonstrated this response, and peptide-stimulated interferon gamma Elispot assays indicated that the response represents a large proportion of the entire CD8+T-lymphocyte SIV- or SHIV-specific immune response of these animals. Knowledge of this epitope and MHC class I allele substantially increases the number of available rhesus monkeys that can be used for testing prototype HIV vaccines in this important animal model.

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.

ABSTRACTSince cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239gag-poland HIV-1 89.6 envelicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.

ABSTRACTIncreasing evidence suggests that the generation of cytotoxic T-lymphocyte (CTL) responses specific for a diversity of viral epitopes will be needed for an effective human immunodeficiency virus type 1 (HIV-1) vaccine. Here, we determine the frequencies of CTL responses specific for the simian immunodeficiency virus Gag p11C and HIV-1 Env p41A epitopes in simian-human immunodeficiency virus (SHIV)-infected and vaccinated rhesus monkeys. The p11C-specific CTL response was high frequency and dominant and the p41A-specific CTL response was low frequency and subdominant in both SHIV-infected monkeys and in monkeys vaccinated with recombinant modified vaccinia virus Ankara vectors expressing these viral antigens. Interestingly, we found that plasmid DNA vaccination led to high-frequency CTL responses specific for both of these epitopes. These data demonstrate that plasmid DNA may be useful in eliciting a broad CTL response against multiple epitopes.

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.

ABSTRACTA tetrameric recombinant major histocompatibility complex (MHC) class II-peptide complex was used to quantitate human immunodeficiency virus type 1 (HIV-1) envelope (Env)-specific CD4+T cells in vaccinated and in simian/human immunodeficiency virus (SHIV)-infected rhesus monkeys. A rhesus monkey MHC class II DR molecule, Mamu-DR*W201, and an HIV-1 Env peptide (p46) were employed to construct this tetrameric complex. A p46-specific proliferative response was seen in sorted, tetramer-binding, but not nonbinding, CD4+T cells, directly demonstrating that this response was mediated by the epitope-specific lymphocytes. Although staining of whole blood from 10 SHIV-infected Mamu-DR*W201+rhesus monkeys failed to demonstrate tetramer-binding CD4+T cells (<0.02%), p46-stimulated peripheral blood mononuclear cells (PBMCs) from 9 of these 10 monkeys had detectable p46 tetramer-binding cells, comprising 0.5 to 15.2% of the CD4+T cells. p46-stimulated PBMCs from 7 of 10 Mamu-DR*W201+monkeys vaccinated with a recombinant canarypox virus–HIV-1 envconstruct also demonstrated p46 tetramer-binding cells, comprising 0.9 to 7.2% of the CD4+T cells. Thus, Env p46-specific CD4+T cells can be detected by tetrameric Mamu-DR*W201–p46 complex staining of PBMCs in both SHIV-infected and vaccinated rhesus monkeys. These epitope-specific cell populations appear to be present in peripheral blood at a very low frequency.

Major histocompatibility class I–peptide tetramer technology and simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys were used to clarify the distribution of acquired immunodeficiency syndrome virus-specific cytotoxic T lymphocytes (CTL) in secondary lymphoid organs and to assess the relationship between these CTL and the extent of viral replication in the various anatomic compartments. SIVmac Gag epitope-specific CD8+ T cells were evaluated in the spleen, bone marrow, tonsils, thymus, and 5 different lymph node compartments of 4 SIVmac-infected rhesus monkeys. The average percentage of CD8+ T lymphocytes that bound this tetramer in all the different lymph node compartments was similar to that in peripheral blood lymphocytes in individual monkeys. The percentage of CD8+ T cells that bound the tetramer in the thymus was uniformly low in the monkeys. However, the percentage of CD8+ T cells that bound the tetramer in bone marrow and spleen was consistently higher than that seen in lymph nodes and peripheral blood. The phenotypic profile of the tetramer-binding CD8+ T lymphocytes in the different lymphoid compartments was similar, showing a high expression of activation-associated adhesion molecules and a low level expression of naive T-cell–associated molecules. Surprisingly, no correlation was evident between the percentage of tetramer-binding CD8+ T lymphocytes and the magnitude of the cell-associated SIV RNA level in each lymphoid compartment of individual monkeys. These studies suggest that a dynamic process of trafficking may obscure the tendency of CTL to localize in particular regional lymph nodes or that some lymphoid organs may provide milieus that are particularly conducive to CTL expansion.

ABSTRACTThe potential contribution of a plasmid DNA construct to vaccine-elicited protective immunity was explored in the simian immunodeficiency virus (SIV)/macaque model of AIDS. Making use of soluble major histocompatibility class I/peptide tetramers and peptide-specific killing assays to monitor CD8+T-lymphocyte responses to a dominant SIV Gag epitope in genetically selected rhesus monkeys, a codon-optimized SIV gagDNA vaccine construct was shown to elicit a high-frequency SIV-specific cytotoxic T-lymphocyte (CTL) response. This CTL response was demonstrable in both peripheral blood and lymph node lymphocytes. Following an intravenous challenge with the highly pathogenic viral isolate SIVsm E660, these vaccinated monkeys developed a secondary CTL response that arose with more rapid kinetics and reached a higher frequency than did the postchallenge CTL response in control plasmid-vaccinated monkeys. While peak plasma SIV RNA levels were comparable in the experimentally and control-vaccinated monkeys during the period of primary infection, the gagplasmid DNA-vaccinated monkeys demonstrated better containment of viral replication by 50 days following SIV challenge. These findings indicate that a plasmid DNA vaccine can elicit SIV-specific CTL responses in rhesus monkeys, and this vaccine-elicited immunity can facilitate the generation of secondary CTL responses and control of viral replication following a pathogenic SIV challenge. These observations suggest that plasmid DNA may prove a useful component of a human immunodeficiency virus type 1 vaccine.

Major histocompatibility class I–peptide tetramer technology and simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys were used to clarify the distribution of acquired immunodeficiency syndrome virus-specific cytotoxic T lymphocytes (CTL) in secondary lymphoid organs and to assess the relationship between these CTL and the extent of viral replication in the various anatomic compartments. SIVmac Gag epitope-specific CD8+T cells were evaluated in the spleen, bone marrow, tonsils, thymus, and 5 different lymph node compartments of 4 SIVmac-infected rhesus monkeys. The average percentage of CD8+T lymphocytes that bound this tetramer in all the different lymph node compartments was similar to that in peripheral blood lymphocytes in individual monkeys. The percentage of CD8+T cells that bound the tetramer in the thymus was uniformly low in the monkeys. However, the percentage of CD8+T cells that bound the tetramer in bone marrow and spleen was consistently higher than that seen in lymph nodes and peripheral blood. The phenotypic profile of the tetramer-binding CD8+T lymphocytes in the different lymphoid compartments was similar, showing a high expression of activation-associated adhesion molecules and a low level expression of naive T-cell–associated molecules. Surprisingly, no correlation was evident between the percentage of tetramer-binding CD8+T lymphocytes and the magnitude of the cell-associated SIV RNA level in each lymphoid compartment of individual monkeys. These studies suggest that a dynamic process of trafficking may obscure the tendency of CTL to localize in particular regional lymph nodes or that some lymphoid organs may provide milieus that are particularly conducive to CTL expansion.

ABSTRACTThe immunogenicity and protective efficacy of a modified vaccinia virus Ankara (MVA) recombinant expressing the simian immunodeficiency virus (SIV) Gag-Pol proteins (MVA-gag-pol) was explored in rhesus monkeys expressing the major histocompatibility complex (MHC) class I allele, MamuA*01. Macaques received four sequential intramuscular immunizations with the MVA-gag-polrecombinant virus or nonrecombinant MVA as a control. Gag-specific cytotoxic T-lymphocyte (CTL) responses were detected in all MVA-gag-pol-immunized macaques by both functional assays and flow cytometric analyses of CD8+T cells that bound a specific MHC complex class I-peptide tetramer, with levels peaking after the second immunization. Following challenge with uncloned SIVsmE660, all macaques became infected; however, viral load set points were lower in MVA-gag-pol-immunized macaques than in the MVA-immunized control macaques. MVA-gag-pol-immunized macaques exhibited a rapid and substantial anamnestic CTL response specific for the p11C, C-M Gag epitope. The level at which CTL stabilized after resolution of primary viremia correlated inversely with plasma viral load set point (P= 0.03). Most importantly, the magnitude of reduction in viremia in the vaccinees was predicted by the magnitude of the vaccine-elicited CTL response prior to SIV challenge.

Nonhuman primates provide valuable animal models for human diseases. However, studies assessing the role of cell-mediated immune responses have been difficult to perform in nonhuman primates. We have shown that CD8+ lymphocyte-mediated immunity in rhesus monkeys can be selectively eliminated using the mouse-human chimeric anti-CD8 monoclonal antibody cM-T807. In vitro, this antibody completely blocked antigen-specific expansion of cytotoxic T cells and decreased major histocompatibility complex class I-restricted, antigen-specific lysis of target cells but did not mediate complement-dependent cell lysis. In vivoadministration of cM-T807 in rhesus monkeys resulted in near total depletion of CD8+ T cells from the blood and lymph nodes for up to 6 weeks. This depletion was not solely complement-dependent and persisted longer in adults than in juveniles. Preservation of B cell and CD4+ T cell function in monkeys depleted of CD8+ lymphocytes was demonstrated by their ability to develop humoral immune responses to the administered chimeric monoclonal antibody. Furthermore, during CD8+ lymphocyte depletion, monkeys developed delayed-type hypersensitivity reactions comprised only of CD4+ T cells but not CD8+ T cells. This CD8+ lymphocyte depletion model should prove useful in defining the role of cell-mediated immune responses in controlling infectious diseases in nonhuman primates.

ABSTRACTTo evaluate the impact of the diversity of antigen recognition by T lymphocytes on disease pathogenesis, we must be able to identify and analyze simultaneously cytotoxic T-lymphocyte (CTL) responses specific for multiple viral epitopes. Many of the studies of the role of CD8+CTLs in AIDS pathogenesis have been done with simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys. These studies have frequently made use of the well-defined SIV Gag CTL epitope p11C,C-M presented to CTL by the HLA-A homologue molecule Mamu-A*01. In the present study we identified and fine mapped two novel Mamu-A*01-restricted CTL epitopes: the SIVmac Pol-derived epitope p68A (STPPLVRLV) and the human immunodeficiency virus type 1 (HIV-1) Env-derived p41A epitope (YAPPISGQI). The frequency of CD8+CTLs specific for the p11C,C-M, p68A, and p41A epitopes was quantitated in the same animals with a panel of tetrameric Mamu-A*01/peptide/ß2m complexes. All SHIV-infected Mamu-A*01+rhesus monkeys tested had a high frequency of SIVmac Gag-specific CTLs to the p11C,C-M epitope. In contrast, only a fraction of the monkeys tested had detectable CTLs specific for the SIVmac Pol p68A and HIV-1 Env p41A epitopes, and these responses were detected at very low frequencies. Thus, the p11C,C-M-specific CD8+CTL response is dominant and the p41A- and p68A-specific CD8+CTL responses are nondominant. These results indicate that CD8+CTL responses to dominant CTL epitopes can be readily quantitated with the tetramer technology; however, CD8+CTL responses to nondominant epitopes, due to the low frequency of these epitope-specific cells, may be difficult to detect and quantitate by this approach.

ABSTRACTEvaluation of human immunodeficiency virus type 1-specific mucosal cytotoxic T lymphocytes can be hampered by limited cell yields from mucosal sites. We sought to characterize virus-specific CD8+T lymphocytes with cytotoxic activity in the male genital tracts of SIVmac-infected rhesus monkeys by using a peptide epitope-specific functional T-cell assay and a tetrameric major histocompatibility complex class I-peptide complex. This tetrameric complex was constructed with the rhesus monkey HLA-A homolog molecule Mamu-A*01 and a dominant-epitope 9-amino-acid fragment of SIVmac Gag (p11C, C-M). The proportion of tetramer-positive CD8+T cells in semen of SIVmac-infected monkeys ranged from 5.9 to 22.0%. By the use of a standard 51Cr release assay, these cells were found to have peptide epitope-specific cytolytic activity after in vitro expansion. Four-color flow-cytometric analysis of these seminal tetramer-positive CD8+T cells demonstrated that they express memory-associated (CD62L-CD45RA-) and activation-associated (CD11a+Fas+HLA-DR+) molecules. The present experiments illustrate the power of tetramer technology for evaluating antigen-specific CD8+T lymphocytes in a mucosal tissue compartment.

ABSTRACTMost studies of human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTL) have been confined to the evaluation of these effector cells in the peripheral blood. What has not been clear is the extent to which CTL activity in the blood actually reflects this effector cell function in the lymph nodes, the major sites of HIV-1 replication. To determine the concordance between CTL activity in lymph nodes and peripheral blood lymphocytes (PBL), CTL specific for simian immunodeficiency virus of macaques (SIVmac) have been characterized in lymph nodes of infected, genetically selected rhesus monkeys by using both Gag peptide-specific functional CTL assays and tetrameric peptide-major histocompatibility complex (MHC) class I molecule complex staining techniques. In studies of six chronically SIVmac-infected rhesus monkeys, Gag epitope-specific functional lytic activity and specific tetrameric peptide-MHC class I staining were readily demonstrated in lymph node T lymphocytes. Although the numbers of tetramer-binding cells in some animals differed from those documented in their PBL, the numbers of tetramer-binding cells from these two different compartments were not statistically different. Phenotypic characterization of the tetramer-binding CD8+lymph node T lymphocytes of the infected monkeys demonstrated a high level of expression of the activation-associated adhesion molecules CD11a and CD49d, the Fas molecule CD95, and MHC class II-DR. These studies documented a low expression of the naive T-cell marker CD45RA and the adhesion molecule CD62L. This phenotypic profile of the tetramer-binding lymph node CD8+T cells was similar to that of tetramer-binding CD8+T cells from PBL. These observations suggest that characterization of AIDS virus-specific CTL activity by sampling of cells in the peripheral blood should provide a reasonable estimation of CTL in an individual’s secondary lymphoid tissue.

To examine whether the mutation of protease in an HIV-1 resistant to a protease inhibitor affects the virus phenotype in vitro, the infectivity of the protease inhibitor-escape-virus was compared to that of the parent virus, In different T-cell lines, the infectivity of the escape virus was impaired by 10-fold compared to the parent virus. MT-4 cell killing by the escape virus, measured using the MTT assay, was much weaker than that by the parent virus The escape virus contained more Unprocessed Pr55^gag than the parent virus. A delayed appearance of mature p24 in cells chronically infected with the escape virus was also noticed by the pulse-chase method. The same findings were obtained using pNL432 (HIV-1 DNA molecular clone) with the same mutation in the protease gene. Despite the lack of a significant difference in virus binding, less unintegrated and integrated DNA was detected in MT-4 cells infected with the escape virus compared to the parent virus. The impaired infectivity of the escape virus may be explained by the inefficient maturation of Gag proteins, due to the mutated protease, which may affect an early step in the virus life cycle. Copyright 1995, 1999 Academic Press

Human immunodeficiency virus type‐1 (HIV‐1) and human T‐cell leukemia virus type‐1 (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.

ABSTRACTThe retroviral attachment (att) sites at viral DNA ends are cis-acting regions essential for proviral integration. To investigate the sequence features of attimportant for human immunodeficiency virus type 1 (HIV-1) integration in vivo, we generated a series of 25 attmutants 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 attsite independently in vivo.

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.

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 μg/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 situhybridization. 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. © 1998 John Wiley & Sons, Ltd.

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 μg/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. © 1998 John Wiley & Sons, Ltd.

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 β2-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)8α/β+ T lymphocyte subset and the percentage of CD8α/β+ 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, CD8α/β+ T cells. Furthermore, the percentage of CD8α/β+ 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.

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.

Unfortunately, the original version of this article was published with error in the materials and methods section.

The tuberculosis (TB) bacillus, Mycobacterium tuberculosis(Mtb), and HIV-1 act synergistically; however, the mechanisms by which Mtb exacerbates HIV-1 pathogenesis are not well known. Using in vitroand ex vivocell culture systems, we show that human M(IL-10) anti-inflammatory macrophages, present in TB-associated microenvironment, produce high levels of HIV-1. In vivo, M(IL-10) macrophages are expanded in lungs of co-infected non-human primates, which correlates with disease severity. Furthermore, HIV-1/Mtb co-infected patients display an accumulation of M(IL-10) macrophage markers (soluble CD163 and MerTK). These M(IL-10) macrophages form direct cell-to-cell bridges, which we identified as tunneling nanotubes (TNTs) involved in viral transfer. TNT formation requires the IL-10/STAT3 signaling pathway, and targeted inhibition of TNTs substantially reduces the enhancement of HIV-1 cell-to-cell transfer and overproduction in M(IL-10) macrophages. Our study reveals that TNTs facilitate viral transfer and amplification, thereby promoting TNT formation as a mechanism to be explored in TB/AIDS potential therapeutics.