Effects of tumor grade and dexamethasone on myeloid cells in patients with glioma.

Efforts to reduce immunosuppression in the solid tumor microenvironment by blocking the recruitment or polarization of tumor associated macrophages (TAM), or myeloid derived suppressor cells (MDSCs), have gained momentum in recent years. Expanding our knowledge of the immune cell types, cytokines, or recruitment factors that are associated with high-grade disease, both within the tumor and in circulation, is critical to identifying novel targets for immunotherapy. Furthermore, a better understanding of how therapeutic regimens, such as Dexamethasone (Dex), chemotherapy, and radiation, impact these factors will facilitate the design of therapies that can be targeted to the appropriate populations and retain efficacy when administered in combination with standard of care regimens. Here we perform quantitative analysis of tissue microarrays made of samples taken from grades I-III astrocytoma and glioblastoma (GBM, grade IV astrocytoma) to evaluate infiltration of myeloid markers CD163, CD68, CD33, and S100A9. Serum, flow cytometric, and Nanostring analysis allowed us to further elucidate the impact of Dex treatment on systemic biomarkers, circulating cells, and functional markers within tumor tissue. We found that common myeloid markers were elevated in Dex-treated grade I astrocytoma and GBM compared to non-neoplastic brain tissue and grade II-III astrocytomas. Cell frequencies in these samples differed significantly from those in Dex-naïve patients in a pattern that depended on tumor grade. In contrast, observed changes in serum chemokines or circulating monocytes were independent of disease state and were due to Dex treatment alone. Furthermore, these changes seen in blood were often not reflected within the tumor tissue. Conclusions: Our findings highlight the importance of considering perioperative treatment as well as disease grade when assessing novel therapeutic targets or biomarkers of disease.

An Uncoupling of Canonical Phenotypic Markers and Functional Potency of Ex Vivo-Expanded Natural Killer Cells.

Recent advances in cellular therapies for patients with cancer, including checkpoint blockade and ex vivo-expanded, tumor-specific T cells, have demonstrated that targeting the immune system is a powerful approach to the elimination of tumor cells. Clinical efforts have also demonstrated limitations, however, including the potential for tumor cell antigenic drift and neoantigen formation, which promote tumor escape and recurrence, as well as rapid onset of T cell exhaustion in vivo. These findings suggest that antigen unrestricted cells, such as natural killer (NK) cells, may be beneficial for use as an alternative to or in combination with T cell based approaches. Although highly effective in lysing transformed cells, to date, few clinical trials have demonstrated antitumor function or persistence of transferred NK cells. Several recent studies describe methods to expand NK cells for adoptive transfer, although the effects of ex vivo expansion are not fully understood. We therefore explored the impact of a clinically validated 12-day expansion protocol using a K562 cell line expressing membrane-bound IL-15 and 4-1BB ligand with high-dose soluble IL-2 on the phenotype and functions of NK cells from healthy donors. Following expansions using this protocol, we found expression of surface proteins that implicate preferential expansion of NK cells that are not fully mature, as is typically associated with highly cytotoxic NK cell subsets. Despite increased expression of markers associated with functional exhaustion in T cells, we found that ex vivo-expanded NK cells retained cytokine production capacity and had enhanced tumor cell cytotoxicity. The preferential expansion of an NK cell subset that is phenotypically immature and functionally pleiotropic suggests that adoptively transferred cells may persist better in vivo when compared with previous methods using this approach. Ex vivo expansion does not quell killer immunoglobulin-like receptor diversity, allowing responsiveness to various factors in vivo that may influence activation and inhibition. Collectively, our data suggest that in addition to robust NK cell expansion that has been described using this method, expanded NK cells may represent an ideal cell therapy that is longer lived, highly potent, and responsive to an array of activating and inhibitory signals.

NKG2D ligand expression in pediatric brain tumors.

Adult brain tumors establish an immunosuppressive tumor microenvironment as a modality of immune escape, with several immunotherapies designed to overcome this barrier. However, the relationship between tumor cells and immune cells in pediatric brain tumor patients is not as well-defined. In this study, we sought to determine whether the model of immune escape observed in adult brain tumors is reflected in patients with pediatric brain tumors by evaluating NKG2D ligand expression on tissue microarrays created from patients with a variety of childhood brain tumor diagnoses, and infiltration of Natural Killer and myeloid cells. We noted a disparity between mRNA and protein expression for the 8 known NKG2D ligands. Surprisingly, high-grade gliomas did not have increased NKG2D ligand expression compared to normal adjacent brain tissue, nor did they have significant myeloid or NK cell infiltration. These data suggest that pediatric brain tumors have reduced NK cell-mediated immune surveillance, and a less immunosuppressive tumor microenvironment as compared to their adult counterparts. These data indicate that therapies aimed to improve NK cell trafficking and functions in pediatric brain tumors may have a greater impact on anti-tumor immune responses and patient survival, with fewer obstacles to overcome.

Flow Cytometry-Based Methods to Characterize Immune Senescence in Nonhuman Primates.

Flow cytometry is an invaluable technique that can be used to phenotypically and functionally characterize immune cell populations ex vivo. This technology has greatly advanced our ability to gain critical insight into age-related changes in immune function, commonly known as immune senescence. Rodents have been traditionally used to investigate the molecular mechanisms of immune senescence because they offer the distinct advantages of an extensive set of reagents, the presence of genetically modified strains, and a short lifespan that allows for longevity studies of short duration. More recently, nonhuman primates (NHPs), and specifically rhesus macaques, have emerged as a leading translational model to study various aspects of human aging. In contrast to rodents, they share significant genetic homology as well as physiological and behavioral characteristics with humans. Furthermore, rhesus macaques are a long-lived outbred species, which makes them an ideal translational model. Therefore, NHPs offer a unique opportunity to carry out mechanistic studies under controlled laboratory conditions (e.g., photoperiod, temperature, diet, and medications) in a species that closely mimics human biology. Moreover similar techniques (e.g., activity recording and MRI) can be used to measure physiological parameters in NHPs, making direct comparisons between NHP and human data sets possible. In addition, the outbred genetics of NHPs enables rigorous validation of research findings that goes beyond proof of principle. Finally, self-selection bias that is often unavoidable in human clinical trials can be completely eliminated with NHP studies. Here we describe flow cytometry-based methods to phenotypically and functionally characterize innate immune cells as well as T and B lymphocyte subsets from isolated peripheral blood mononuclear cells (PBMC) in rhesus macaques.

Abortive intrabronchial infection of rhesus macaques with varicella-zoster virus provides partial protection against simian varicella virus challenge.

UNLABELLED: Varicella-zoster virus (VZV) is a human neurotropic alphaherpesvirus and the etiological agent of varicella (chickenpox) and herpes zoster (HZ, shingles). Previously, inoculation of monkeys via the subcutaneous, intratracheal, intravenous, or oral-nasal-conjunctival routes did not recapitulate all the hallmarks of VZV infection, including varicella, immunity, latency, and reactivation. Intrabronchial inoculation of rhesus macaques (RMs) with simian varicella virus (SVV), a homolog of VZV, recapitulates virologic and immunologic hallmarks of VZV infection in humans. Given that VZV is acquired primarily via the respiratory route, we investigated whether intrabronchial inoculation of RMs with VZV would result in a robust model. Despite the lack of varicella and viral replication in either the lungs or whole blood, all four RMs generated an immune response characterized by the generation of VZV-specific antibodies and T cells. Two of 4 VZV-inoculated RMs were challenged with SVV to determine cross-protection. VZV-immune RMs displayed no varicella rash and had lower SVV viral loads and earlier and stronger humoral and cellular immune responses than controls. In contrast to the results for SVV DNA, no VZV DNA was detected in sensory ganglia at necropsy. In summary, following an abortive VZV infection, RMs developed an adaptive immune response that conferred partial protection against SVV challenge. These data suggest that a replication-incompetent VZV vaccine that does not establish latency may provide sufficient protection against VZV disease and that VZV vaccination of RMs followed by SVV challenge provides a model to evaluate new vaccines and therapeutics against VZV. IMPORTANCE: Although VZV vaccine strain Oka is attenuated, it can cause mild varicella, establish latency, and in rare cases, reactivate to cause herpes zoster (HZ). Moreover, studies suggest that the HZ vaccine (Zostavax) only confers short-lived immunity. The development of more efficacious vaccines would be facilitated by a robust animal model of VZV infection. The data presented in this report show that intrabronchial inoculation of rhesus macaques (RMs) with VZV resulted in an abortive VZV infection. Nevertheless, all animals generated a humoral and cellular immune response that conferred partial cross-protection against simian varicella virus (SVV) challenge. Additionally, VZV DNA was not detected in the sensory ganglia, suggesting that viremia might be required for the establishment of latency. Therefore, VZV vaccination of RMs followed by SVV challenge is a model that will support the development of vaccines that boost protective T cell responses against VZV.

Immune evasion mediated by tumor-derived lactate dehydrogenase induction of NKG2D ligands on myeloid cells in glioblastoma patients.

Myeloid cells are key regulators of the tumor microenvironment, governing local immune responses. Here we report that tumor-infiltrating myeloid cells and circulating monocytes in patients with glioblastoma multiforme (GBM) express ligands for activating the Natural killer group 2, member D (NKG2D) receptor, which cause down-regulation of NKG2D on natural killer (NK) cells. Tumor-infiltrating NK cells isolated from GBM patients fail to lyse NKG2D ligand-expressing tumor cells. We demonstrate that lactate dehydrogenase (LDH) isoform 5 secreted by glioblastoma cells induces NKG2D ligands on monocytes isolated from healthy individuals. Furthermore, sera from GBM patients contain elevated amounts of LDH, which correlate with expression of NKG2D ligands on their autologous circulating monocytes. NKG2D ligands also are present on circulating monocytes isolated from patients with breast, prostate, and hepatitis C virus-induced hepatocellular carcinomas. Together, these findings reveal a previously unidentified immune evasion strategy whereby tumors produce soluble factors that induce NKG2D ligands on myeloid cells, subverting antitumor immune responses.

Intrabronchial infection of rhesus macaques with simian varicella virus results in a robust immune response in the lungs.

UNLABELLED: Varicella-zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (shingles). Primary VZV infection is believed to occur via the inhalation of virus either in respiratory droplets or from shedding varicella lesions or by direct contact with infectious vesicular fluid. However, the ensuing immune response in the lungs remains incompletely understood. We have shown that intrabronchial inoculation of rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we performed an in-depth analysis of the host immune response to acute SVV infection in the lungs and peripheral blood. We report that acute SVV infection results in a robust innate immune response in the lungs, characterized by the production of inflammatory cytokines, chemokines, and growth factors as well as an increased frequency of plasmacytoid dendritic cells (DCs) that corresponded with alpha interferon (IFN-α) production and a rapid decrease in viral loads in the lungs. This is followed by T and B cell proliferation, antibody production, T cell differentiation, and cytokine production, which correlate with the complete cessation of viral replication. Although terminally differentiated CD8 T cells became the predominant T cell population in bronchoalveolar lavage cells, a higher percentage of CD4 T cells were SVV specific, which suggests a critical role for these cells in the resolution of primary SVV infection in the lungs. Given the homology between SVV and VZV, our data provide insight into the immune response to VZV within the lung. IMPORTANCE: Although primary VZV infection occurs primarily via the respiratory route, the host response in the lungs and its contribution to the cessation of viral replication and establishment of latency remain poorly understood. The difficulty in accessing lung tissue and washes from individuals infected with VZV has hampered efforts to address this knowledge gap. SVV infection of rhesus macaques is an important model of VZV infection of humans; therefore, we utilized this animal model to gain a comprehensive view of the kinetics of the immune response to SVV in the lung and its relationship to the resolution of acute infection in respiratory tissues. These data not only advance our understanding of host immunity to VZV, a critical step in developing new vaccines, but also provide additional insight into immunity to respiratory pathogens.

Chronic ethanol consumption modulates growth factor release, mucosal cytokine production, and microRNA expression in nonhuman primates.

BACKGROUND: Chronic alcohol consumption has been associated with enhanced susceptibility to both systemic and mucosal infections. However, the exact mechanisms underlying this enhanced susceptibility remain incompletely understood. METHODS: Using a nonhuman primate model of ethanol (EtOH) self-administration, we examined the impact of chronic alcohol exposure on immune homeostasis, cytokine, and growth factor production in peripheral blood, lung, and intestinal mucosa following 12 months of chronic EtOH exposure. RESULTS: EtOH exposure inhibited activation-induced production of growth factors hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), and vascular-endothelial growth factor (VEGF) by peripheral blood mononuclear cells (PBMC). Moreover, EtOH significantly reduced the frequency of colonic Th1 and Th17 cells in a dose-dependent manner. In contrast, we did not observe differences in lymphocyte frequency or soluble factor production in the lung of EtOH-consuming animals. To uncover mechanisms underlying reduced growth factor and Th1/Th17 cytokine production, we compared expression levels of microRNAs in PBMC and intestinal mucosa. Our analysis revealed EtOH-dependent up-regulation of distinct microRNAs in affected tissues (miR-181a and miR-221 in PBMC; miR-155 in colon). Moreover, we were able to detect reduced expression of the transcription factors STAT3 and ARNT, which regulate expression of VEGF, G-CSF, and HGF and contain targets for these microRNAs. To confirm and extend these observations, PBMC were transfected with either mimics or antagomirs of miR-181 and miR-221, and protein levels of the transcription factors and growth factors were determined. Transfection of microRNA mimics led to a reduction in both STAT3/ARNT as well as VEGF/HGF/G-CSF levels. The opposite outcome was observed when microRNA antagomirs were transfected. CONCLUSIONS: Chronic EtOH consumption significantly disrupts both peripheral and mucosal immune homeostasis, and this dysregulation may be mediated by changes in microRNA expression.

Bacterial artificial chromosome derived simian varicella virus is pathogenic in vivo.

BACKGROUND: Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that infects humans and results in chickenpox and herpes zoster. A number of VZV genes remain functionally uncharacterized and since VZV is an obligate human pathogen, rigorous evaluation of VZV mutants in vivo remains challenging. Simian varicella virus (SVV) is homologous to VZV and SVV infection of rhesus macaques (RM) closely mimics VZV infection of humans. Recently the SVV genome was cloned as a bacterial artificial chromosome (BAC) and BAC-derived SVV displayed similar replication kinetics as wild-type (WT) SVV in vitro. METHODS: RMs were infected with BAC-derived SVV or WT SVV at 4x10(5) PFU intrabronchially (N=8, 4 per group, sex and age matched). We collected whole blood (PBMC) and bronchoalveolar lavage (BAL) at various days post-infection (dpi) and sensory ganglia during latent infection (>84 dpi) at necropsy and compared disease progression, viral replication, immune response and the establishment of latency. RESULTS: Viral replication kinetics and magnitude in bronchoalveolar lavage cells and whole blood as well as rash severity and duration were similar in RMs infected with SVV BAC or WT SVV. Moreover, SVV-specific B and T cell responses were comparable between BAC and WT-infected animals. Lastly, we measured viral DNA in sensory ganglia from both cohorts of infected RMs during latent infection. CONCLUSIONS: SVV BAC is as pathogenic and immunogenic as WT SVV in vivo. Thus, the SVV BAC genetic system combined with the rhesus macaque animal model can further our understanding of viral ORFs important for VZV pathogenesis and the development of second-generation vaccines.

Chikungunya virus infection results in higher and persistent viral replication in aged rhesus macaques due to defects in anti-viral immunity.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne Alphavirus that causes a clinical disease involving fever, myalgia, nausea and rash. The distinguishing feature of CHIKV infection is the severe debilitating poly-arthralgia that may persist for several months after viral clearance. Since its re-emergence in 2004, CHIKV has spread from the Indian Ocean region to new locations including metropolitan Europe, Japan, and even the United States. The risk of importing CHIKV to new areas of the world is increasing due to high levels of viremia in infected individuals as well as the recent adaptation of the virus to the mosquito species Aedes albopictus. CHIKV re-emergence is also associated with new clinical complications including severe morbidity and, for the first time, mortality. In this study, we characterized disease progression and host immune responses in adult and aged Rhesus macaques infected with either the recent CHIKV outbreak strain La Reunion (LR) or the West African strain 37997. Our results indicate that following intravenous infection and regardless of the virus used, Rhesus macaques become viremic between days 1-5 post infection. While adult animals are able to control viral infection, aged animals show persistent virus in the spleen. Virus-specific T cell responses in the aged animals were reduced compared to adult animals and the B cell responses were also delayed and reduced in aged animals. Interestingly, regardless of age, T cell and antibody responses were more robust in animals infected with LR compared to 37997 CHIKV strain. Taken together these data suggest that the reduced immune responses in the aged animals promotes long-term virus persistence in CHIKV-LR infected Rhesus monkeys.

Genome-wide analysis of T cell responses during acute and latent simian varicella virus infections in rhesus macaques.

Varicella zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (HZ [shingles]). Clinical observations suggest that VZV-specific T cell immunity plays a more critical role than humoral immunity in the prevention of VZV reactivation and development of herpes zoster. Although numerous studies have characterized T cell responses directed against select VZV open reading frames (ORFs), a comprehensive analysis of the T cell response to the entire VZV genome has not yet been conducted. We have recently shown that intrabronchial inoculation of young rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we characterized the specificity of T cell responses during acute and latent SVV infection. Animals generated a robust and broad T cell response directed against both structural and nonstructural viral proteins during acute infection in bronchoalveolar lavage (BAL) fluid and peripheral blood. During latency, T cell responses were detected only in the BAL fluid and were lower and more restricted than those observed during acute infection. Interestingly, we identified a small set of ORFs that were immunogenic during both acute and latent infection in the BAL fluid. Given the close genome relatedness of SVV and VZV, our studies highlight immunogenic ORFs that may be further investigated as potential components of novel VZV vaccines that specifically boost T cell immunity.

Age and immune status of rhesus macaques impact simian varicella virus gene expression in sensory ganglia.

Simian varicella virus (SVV) infection of rhesus macaques (RMs) recapitulates the hallmarks of varicella-zoster virus (VZV) infection of humans, including the establishment of latency within the sensory ganglia. Various factors, including age and immune fitness, influence the outcome of primary VZV infection, as well as reactivation resulting in herpes zoster (HZ). To increase our understanding of the role of lymphocyte subsets in the establishment of viral latency, we analyzed the latent SVV transcriptome in juvenile RMs depleted of CD4 T, CD8 T, or CD20 B lymphocytes during acute infection. We have previously shown that SVV latency in sensory ganglia of nondepleted juvenile RMs is associated with a limited transcriptional profile. In contrast, CD4 depletion during primary infection resulted in the failure to establish a characteristic latent viral transcription profile in sensory ganglia, where we detected 68 out of 69 SVV-encoded open reading frames (ORFs). CD-depleted RMs displayed a latent transcriptional profile that included additional viral transcripts within the core region of the genome not detected in control RMs. The latent transcriptome of CD20-depleted RMs was comparable to the latent transcription in the sensory ganglia of control RMs. Lastly, we investigated the impact of age on the establishment of SVV latency. SVV gene expression was more active in ganglia from two aged RMs than in ganglia from juvenile RMs, with 25 of 69 SVV transcripts detected. Therefore, immune fitness at the time of infection modulates the establishment and/or maintenance of SVV latency.

Antibodies are necessary for rVSV/ZEBOV-GP-mediated protection against lethal Ebola virus challenge in nonhuman primates.

Ebola viruses cause hemorrhagic disease in humans and nonhuman primates with high fatality rates. These viruses pose a significant health concern worldwide due to the lack of approved therapeutics and vaccines as well as their potential misuse as bioterrorism agents. Although not licensed for human use, recombinant vesicular stomatitis virus (rVSV) expressing the filovirus glycoprotein (GP) has been shown to protect macaques from Ebola virus and Marburg virus infections, both prophylactically and postexposure in a homologous challenge setting. However, the immune mechanisms of protection conferred by this vaccine platform remain poorly understood. In this study, we set out to investigate the role of humoral versus cellular immunity in rVSV vaccine-mediated protection against lethal Zaire ebolavirus (ZEBOV) challenge. Groups of cynomolgus macaques were depleted of CD4+ T, CD8+ T, or CD20+ B cells before and during vaccination with rVSV/ZEBOV-GP. Unfortunately, CD20-depleted animals generated a robust IgG response. Therefore, an additional group of vaccinated animals were depleted of CD4+ T cells during challenge. All animals were subsequently challenged with a lethal dose of ZEBOV. Animals depleted of CD8+ T cells survived, suggesting a minimal role for CD8+ T cells in vaccine-mediated protection. Depletion of CD4+ T cells during vaccination caused a complete loss of glycoprotein-specific antibodies and abrogated vaccine protection. In contrast, depletion of CD4+ T cells during challenge resulted in survival of the animals, indicating a minimal role for CD4+ T-cell immunity in rVSV-mediated protection. Our results suggest that antibodies play a critical role in rVSV-mediated protection against ZEBOV.

Animal models of varicella zoster virus infection.

Primary infection with varicella zoster virus (VZV) results in varicella (chickenpox) followed by the establishment of latency in sensory ganglia. Declining T cell immunity due to aging or immune suppressive treatments can lead to VZV reactivation and the development of herpes zoster (HZ, shingles). HZ is often associated with significant morbidity and occasionally mortality in elderly and immune compromised patients. There are currently two FDA-approved vaccines for the prevention of VZV: Varivax® (for varicella) and Zostavax® (for HZ). Both vaccines contain the live-attenuated Oka strain of VZV. Although highly immunogenic, a two-dose regimen is required to achieve a 99% seroconversion rate. Zostavax vaccination reduces the incidence of HZ by 51% within a 3-year period, but a significant reduction in vaccine-induced immunity is observed within the first year after vaccination. Developing more efficacious vaccines and therapeutics requires a better understanding of the host response to VZV. These studies have been hampered by the scarcity of animal models that recapitulate all aspects of VZV infections in humans. In this review, we describe different animal models of VZV infection as well as an alternative animal model that leverages the infection of Old World macaques with the highly related simian varicella virus (SVV) and discuss their contributions to our understanding of pathogenesis and immunity during VZV infection.

Attenuation of the adaptive immune response in rhesus macaques infected with simian varicella virus lacking open reading frame 61.

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that causes chickenpox during primary infection and establishes latency in sensory ganglia. Infection of rhesus macaques (RM) with the homologous simian varicella virus (SVV) recapitulates hallmarks of VZV infection. We have shown that an antisense transcript of SVV open reading frame 61 (ORF61), a viral transactivator, was detected most frequently in latently infected RM sensory ganglia. In this study, we compared disease progression, viral replication, immune response, and the establishment of latency following intrabronchial infection with a recombinant SVV lacking ORF61 (SVVΔORF61) to those following infection with wild-type (WT) SVV. Varicella severity and viral latency within sensory ganglia were comparable in RMs infected with SVVΔORF61 and WT SVV. In contrast, viral loads, B and T cell responses, and plasma inflammatory cytokine levels were decreased in RMs infected with SVVΔORF61. To investigate the mechanisms underlying the reduced adaptive immune response, we compared acute SVV gene expression, frequency and proliferation of dendritic cell (DC) subsets, and the expression of innate antiviral genes in bronchoalveolar lavage (BAL) samples. The abundance of SVV transcripts in all kinetic classes was significantly decreased in RMs infected with SVVΔORF61. In addition, we detected a higher frequency and proliferation of plasmacytoid dendritic cells in BAL fluid at 3 days postinfection in RMs infected with SVVΔORF61, which was accompanied by a slight increase in type I interferon gene expression. Taken together, our data suggest that ORF61 plays an important role in orchestrating viral gene expression in vivo and interferes with the host antiviral interferon response.

Age-dependent changes in innate immune phenotype and function in rhesus macaques (Macaca mulatta).

Aged individuals are more susceptible to infections due to a general decline in immune function broadly referred to as immune senescence. While age-related changes in the adaptive immune system are well documented, aging of the innate immune system remains less well understood, particularly in nonhuman primates. A more robust understanding of age-related changes in innate immune function would provide mechanistic insight into the increased susceptibility of the elderly to infection. Rhesus macaques have proved a critical translational model for aging research, and present a unique opportunity to dissect age-dependent modulation of the innate immune system. We examined age-related changes in: (i) innate immune cell frequencies; (ii) expression of pattern recognition receptors (PRRs) and innate signaling molecules; (iii) cytokine responses of monocytes and dendritic cells (DC) following stimulation with PRR agonists; and (iv) plasma cytokine levels in this model. We found marked changes in both the phenotype and function of innate immune cells. This included an age-associated increased frequency of myeloid DC (mDC). Moreover, we found toll-like receptor (TLR) agonists lipopolysaccharide (TLR4), fibroblast stimulating ligand-1 (TLR2/6), and ODN2006 (TLR7/9) induced reduced cytokine responses in aged mDC. Interestingly, with the exception of the monocyte-derived TNFα response to LPS, which increased with age, TNFα, IL-6, and IFNα responses declined with age. We also found that TLR4, TLR5, and innate negative regulator, sterile alpha and TIR motif containing protein (SARM), were all expressed at lower levels in young animals. By contrast, absent in melanoma 2 and retinoic acid-inducible gene I expression was lowest in aged animals. Together, these observations indicate that several parameters of innate immunity are significantly modulated by age and contribute to differential immune function in aged macaques.

Increased viral loads and exacerbated innate host responses in aged macaques infected with the 2009 pandemic H1N1 influenza A virus.

In contrast to seasonal influenza virus infections, which typically cause significant morbidity and mortality in the elderly, the 2009 H1N1 virus caused severe infection in young adults. This phenomenon was attributed to the presence of cross-protective antibodies acquired by older individuals during previous exposures to H1N1 viruses. However, this hypothesis could not be empirically tested. To address this question, we compared viral replication and the development of the immune response in naïve young adult and aged female rhesus macaques infected with A/California/04/2009 H1N1 (CA04) virus. We show higher viral loads in the bronchoalveolar lavage (BAL) fluid and nasal and ocular swabs in aged animals, suggesting increased viral replication in both the lower and upper respiratory tracts. T cell proliferation was higher in the BAL fluid but delayed and reduced in peripheral blood in aged animals. This delay in proliferation correlated with a reduced frequency of effector CD4 T cells in old animals. Aged animals also mobilized inflammatory cytokines to higher levels in the BAL fluid. Finally, we compared changes in gene expression using microarray analysis of BAL fluid samples. Our analyses revealed that the largest difference in host response between aged and young adult animals was detected at day 4 postinfection, with a significantly higher induction of genes associated with inflammation and the innate immune response in aged animals. Overall, our data suggest that, in the absence of preexisting antibodies, CA04 infection in aged macaques is associated with changes in innate and adaptive immune responses that were shown to correlate with increased disease severity in other respiratory disease models.

Improving immunity in the elderly: current and future lessons from nonhuman primate models.

The immune system must overcome daily challenges from pathogens to protect the body from infection. The success of the immune response to infection relies on the ability to sense and evaluate microbial threats and organize their elimination, while limiting damage to host tissues. This delicate balance is achieved through coordinated action of the innate and adaptive arms of the immune system. Aging results in several structural and functional changes in the immune system, often described under the umbrella term "immune senescence". Age-related changes affect both the innate and adaptive arms of the immune system and are believed to result in increased susceptibility and severity of infectious diseases, which is further exacerbated by reduced vaccine efficacy in the elderly. Therefore, multiple strategies to improve immune function in the aged are being investigated. Traditionally, studies on immune senescence are conducted using inbred specific pathogen free (SPF) rodents. This animal model has provided invaluable insight into the mechanisms of aging. However, the limited genetic heterogeneity and the SPF status of this model restrict the successful transfer of immunological discoveries between murine models and the clinical setting. More recently, nonhuman primates (NHPs) have emerged as a leading translational model to investigate immune senescence and to test interventions aimed at delaying/reversing age-related changes in immune function. In this article, we review and summarize advances in immuno-restorative approaches investigated in the NHP model system and discuss where the NHP model can support the development of novel therapeutics.

CD4 T cell immunity is critical for the control of simian varicella virus infection in a nonhuman primate model of VZV infection.

Primary infection with varicella zoster virus (VZV) results in varicella (more commonly known as chickenpox) after which VZV establishes latency in sensory ganglia. VZV can reactivate to cause herpes zoster (shingles), a debilitating disease that affects one million individuals in the US alone annually. Current vaccines against varicella (Varivax) and herpes zoster (Zostavax) are not 100% efficacious. Specifically, studies have shown that 1 dose of varivax can lead to breakthrough varicella, albeit rarely, in children and a 2-dose regimen is now recommended. Similarly, although Zostavax results in a 50% reduction in HZ cases, a significant number of recipients remain at risk. To design more efficacious vaccines, we need a better understanding of the immune response to VZV. Clinical observations suggest that T cell immunity plays a more critical role in the protection against VZV primary infection and reactivation. However, no studies to date have directly tested this hypothesis due to the scarcity of animal models that recapitulate the immune response to VZV. We have recently shown that SVV infection of rhesus macaques models the hallmarks of primary VZV infection in children. In this study, we used this model to experimentally determine the role of CD4, CD8 and B cell responses in the resolution of primary SVV infection in unvaccinated animals. Data presented in this manuscript show that while CD20 depletion leads to a significant delay and decrease in the antibody response to SVV, loss of B cells does not alter the severity of varicella or the kinetics/magnitude of the T cell response. Loss of CD8 T cells resulted in slightly higher viral loads and prolonged viremia. In contrast, CD4 depletion led to higher viral loads, prolonged viremia and disseminated varicella. CD4 depleted animals also had delayed and reduced antibody and CD8 T cell responses. These results are similar to clinical observations that children with agammaglobulinemia have uncomplicated varicella whereas children with T cell deficiencies are at increased risk of progressive varicella with significant complications. Moreover, our studies indicate that CD4 T cell responses to SVV play a more critical role than antibody or CD8 T cell responses in the control of primary SVV infection and suggest that one potential mechanism for enhancing the efficacy of VZV vaccines is by eliciting robust CD4 T cell responses.

Immune senescence in aged nonhuman primates.

Aging is accompanied by a general dysregulation in immune system function, commonly referred to as 'immune senescence'. This progressive deterioration affects both innate and adaptive immunity, although accumulating evidence indicates that the adaptive arm of the immune system may exhibit more profound changes. Most of our current understanding of immune senescence stems from clinical and rodent studies. More recently, the use of nonhuman primates (NHPs) to investigate immune senescence and test interventions aimed at delaying/reversing age-related changes in immune function has dramatically increased. These studies have been greatly facilitated by several key advances in our understanding of the immune system of old world monkeys, specifically the rhesus macaques. In this review we describe the hallmarks of immune senescence in this species and compare them to those described in humans. We also discuss the impact of immune senescence on the response to vaccination and the efficacy of immuno-restorative interventions investigated in this model system.