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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a global economic and health crisis. Recently, plasma levels of galectin-9 (Gal-9), a β-galactoside-binding lectin involved in immune regulation and viral immunopathogenesis, were reported to be elevated in the setting of severe COVID-19 disease. However, the impact of Gal-9 on SARS-CoV-2 infection and immunopathology remained to be elucidated. In this study, we demonstrate that Gal-9 treatment potently enhances SARS-CoV-2 replication in human airway epithelial cells (AECs), including immortalized AECs and primary AECs cultured at the air-liquid interface. Gal-9-glycan interactions promote SARS-CoV-2 attachment and entry into AECs in an angiotensin-converting enzyme 2 (ACE2)-dependent manner, enhancing the binding of the viral spike protein to ACE2. Transcriptomic analysis revealed that Gal-9 and SARS-CoV-2 infection synergistically induced the expression of key pro-inflammatory programs in AECs, including the IL-6, IL-8, IL-17, EIF2, and TNFα signaling pathways. Our findings suggest that manipulation of Gal-9 should be explored as a therapeutic strategy for SARS-CoV-2 infection.

Despite viral suppression with antiretroviral therapy (ART), people with human immunodeficiency virus (HIV) continue to experience central nervous system (CNS) complications, primarily in the form of mild cognitive impairment and mental health disorders (eg, depression, anxiety, other neuropsychiatric problems). The multifactorial pathogenesis and heterogeneity of mechanisms likely underlying CNS complications must be addressed in the development of preventive interventions and effective treatments. The biotyping approach has previously been useful to define phenotypes of other CNS diseases based on underlying mechanisms and could be translated to the field of neuroHIV. The purpose of the Biotype Workshop series, and the Virology, Immunology and Neuropathology Working Group in particular, is to capitalize on current and new technologies and guide future research efforts using the wealth of available immunological, virologic, and neuropathological data collected from people with HIV on and off ART.

HIV-infected individuals have increased risk for cardiovascular disease (CVD) despite suppressive antiretroviral therapy (ART). This is likely a result of persistent immune activation and systemic inflammation. Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and may drive inflammation contributing to CVD. EVs were characterized in plasma from 74 HIV-infected individuals on combination antiretroviral therapy (cART) and 64 HIV-uninfected controls with paired carotid intima-media thickness (cIMT) assessment. EVs were profiled with markers reflecting lymphoid, myeloid, and endothelial origin. Seventeen plasma inflammatory biomarkers were also assessed. Human umbilical vein endothelial cell (HUVEC) apoptosis was quantified after EV exposure. A significant correlation was observed in HIV-infected participants between cIMT and EVs expressing CD16, and the monocyte-related markers CD4, CD14, and CX3CR1 showed a similar but nonsignificant association with cIMT. No significant correlation between cIMT measurements from HIV-uninfected individuals and EVs was observed. Levels of serum amyloid A, C-reactive protein, and myeloperoxidase significantly correlated with CD14+, CD16+, and CX3CR1+ EVs. No correlation was noted between cIMT and soluble inflammatory markers. HUVECs showed increased necrosis after exposure to the EV-containing fraction of plasma derived from HIV-infected individuals compared to uninfected controls. Our study reveals that EVs expressing monocyte markers correlated with cIMT in HIV-infected individuals on cART. Moreover, EV fractions derived from HIV-infected individuals lead to greater endothelial cell death via necrotic pathways. Collectively, EVs have potential as biomarkers of and therapeutic targets in the pathogenesis of CVD in the setting of treated HIV disease. IMPORTANCE HIV-infected individuals have a 2-fold-increased risk of cardiovascular disease compared with the general population, yet the mechanisms underlying this comorbidity are unclear. Extracellular vesicles have emerged as important mediators in cell-cell communication and, given what we know of their biology, may drive inflammation contributing to cardiovascular disease in this vulnerable population.

Cognitive post-acute sequelae of SARS-CoV-2 (PASC) can occur after mild COVID-19. Detailed clinical characterizations may inform pathogenesis. We evaluated 22 adults reporting cognitive PASC and 10 not reporting cognitive symptoms after mild SARS-CoV-2 infection through structured interviews, neuropsychological testing, and optional cerebrospinal fluid (CSF) evaluations (53%). Delayed onset of cognitive PASC occurred in 43% and associated with younger age. Cognitive PASC participants had a higher number of pre-existing cognitive risk factors (2.5 vs. 0; p = 0.03) and higher proportion with abnormal CSF findings (77% vs. 0%; p = 0.01) versus controls. Cognitive risk factors and immunologic mechanisms may contribute to cognitive PASC pathogenesis.

BackgroundNeurocognitive impairment remains prevalent in people with HIV (PWH) despite long term virological suppression by antiretroviral therapy (ART) regimens. Systemic and neuro-inflammatory processes are suggested to contribute to the complex pathology leading to cognitive impairment in this population, yet the underlying mechanisms remain unresolved. Extracellular vesicles (EVs) play a central role in intracellular communication and have emerged as key modulators of immunological and inflammatory responses. In this report, we examined the impact of EVs in PWH experiencing cognitive deficits to determine their relevance in HIV associated neuropathology.MethodsEV phenotypes were measured in plasma samples from 108 PWH with either cognitive impairment (CI, n=92) or normal cognition (NC, n=16) by flow cytometry. Matched cerebrospinal fluid (CSF)-derived EVs were similarly profiled from a subgroup of 84 individuals who underwent a lumbar puncture. Peripheral blood mononuclear cells were assayed by flow cytometry to measure monocyte frequencies in a subset of 32 individuals.ResultsPlasma-EVs expressing CD14, CD16, CD192, C195, and GFAP were significantly higher in HIV-infected individuals with cognitive impairment compared to individuals with normal cognition. Increased CSF-EVs expressing GFAP and CD200 were found in the cognitive impairment group compared to the normal cognition group. Frequencies of patrolling monocytes correlated with plasma-EVs expressing CD14, CD66b, MCSF, MAP2, and GFAP. Frequencies of CD195 expression on monocytes correlated positively with plasma-EVs expressing CD41a, CD62P, and CD63. Expression of CD163 on monocytes correlated positively with CSF-EVs expressing GFAP and CD200. Finally, the expression of CD192 on total monocytes correlated with CSF-EVs expressing CD200, CD62P, and CD63.ConclusionsEVs expressing monocyte activation and neuronal markers associated with HIV associated cognitive impairment, suggesting that distinct EV subsets may serve as novel biomarkers of neuronal injury in HIV infection. Further circulating platelet EV levels were linked to monocyte activation indicating a potential novel interaction in the pathogenesis of HIV-related cognitive impairment.

BackgroundPeople with HIV (PWH) on antiretroviral therapy (ART) still experience an increased risk of morbidity and mortality, presumably driven by chronic inflammation, yet predictors of discrete or combinatorial outcomes remain unclear. Galectin-9 (Gal-9), a driver of both inflammatory and immunosuppressive responses, has been associated with HIV disease progression and multimorbidity.ObjectiveTo determine whether plasma Gal-9 levels are associated with the occurrence of specific non-AIDS events (NAEs) in PWH initiating ART.DesignWe performed a nested case-control study of PWH enrolled from 2001 to 2009 and evaluated pre-ART (66 cases, 97 controls), a year post-ART (112 cases, 211 controls), and immediately preceding an event (89 cases, 162 controls). Events included myocardial infarction/stroke, malignancy, serious bacterial infection, or death.MethodsPlasma Gal-9 levels were assessed by ELISA. Conditional logistic regression assessed associations with NAEs and Spearman's correlations compared Gal-9 with other previously assessed biomarkers.ResultsNAEs occurred at a median of 2.8 years (1.7-4.6) after ART initiation. Higher Gal-9 levels were associated with increased risk of NAEs at year 1 and preevent [odds ratio (OR) per 1 interquartile range = 1.4-1.6; all P

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) develops in 1-5% of HTLV-1-infected individuals. Previous studies by us and others have shown that the expression of negative immune checkpoint receptors (NCRs) is significantly increased on CD8 T cells in various chronic viral infections and are associated with poor anti-viral immunity. We have previously identified the differential expression of NCRs on CD8 T cells in blood from patients with HAM/TSP and in central nervous system (CNS) tissues of HTLV-1 infected humanized mice and defined the association with neurological complications. In this study, we determined the co-expression patterns of several key NCRs (PD-1, TIGIT, TIM-3, and LAG-3) and their cognate ligands in HTLV-1 infection and assessed how combination strategies targeting these pathways would impact HTLV-1-specific CD8 T-cell effector functions as an approach to reduce CNS disease outcomes. We found that global CD8 T cells from HAM/TSP patients co-express multiple NCRs at significantly higher frequencies than asymptomatic carriers (AC). Moreover, NCR ligands (PVR and PD-LI) on both plasmacytoid and myeloid dendritic cells were also expressed at higher frequencies in HAM/TSP compared to AC. In both AC and HAM/TSP subjects, combination dual PD-L1/TIGIT or triple PD-L1/TIGIT/TIM-3 blockade with monoclonal antibodies resulted in increases in intracellular cytokine expression in CD8 T cells after virus stimulation, particularly CD107a, a marker of degranulation, and TNF-α, a key cytokine that can directly inhibit viral replication. Interestingly, almost all blockade combinations resulted in reduced IL-2+ HTLV-1-specific CD8 T cell frequencies in HAM/TSP subjects, but not in AC. These results define a novel combinatorial NCR immunotherapeutic blockade strategy to reduce HAM/TSP disease burden.

ObjectivesThe aim of this study was to examine the relationship between gut microbial communities in HIV-infected individuals on suppressive antiretroviral therapy (cART), and the peripheral HIV-Gag-specific CD8 T-cell responses before and after ex-vivo immune checkpoint blockade (ICB).DesignThirty-four HIV-seropositive, 10 HIV-seronegative and 12 HIV-seropositive receiving faecal microbiota transplant (FMT) participants were included. Gut microbial communities, peripheral and gut associated negative checkpoint receptors (NCRs) and peripheral effector functions were assessed.MethodsBacterial 16s rRNA sequencing for gut microbiome study and flow-based assays for peripheral and gut NCR and their cognate ligand expression, including peripheral HIV-Gag-specific CD8 T-cell responses before and after ex-vivo anti-PD-L1 and anti-TIGIT ICB were performed.ResultsFusobacteria abundance was significantly higher in HIV-infected donors compared to uninfected controls. In HIV-infected participants receiving Fusobacteria-free FMT, Fusobacteria persisted up to 24 weeks in stool post FMT. PD-1 TIGIT and their ligands were expanded in mucosal vs. peripheral T cells and dendritic cells, respectively. PD-L1 and TIGIT blockade significantly increased the magnitude of peripheral anti-HIV-Gag-specific CD8 T-cell responses. Higher gut Fusobacteria abundance was associated with lower magnitude of peripheral IFN-γ+ HIV-Gag-specific CD8 T-cell responses following ICB.ConclusionThe gut colonization of Fusobacteria in HIV infection is persistent and may influence anti-HIV T-cell immunity to PD-1 or TIGIT blockade. Strategies modulating Fusobacteria colonization may elicit a favourable mucosal immune landscape to enhance the efficacy of ICB for HIV cure.

ObjectiveInflammation may contribute to brain white matter health in people living with HIV who report cognitive symptoms despite adherence to combination antiretroviral therapy and viral suppression. We explored relationships between diffusion tensor imaging (DTI) metrics of white matter, plasma biomarkers of immune activation, and cognitive function in the HIV-infected population.DesignRetrospective study of older adults living with HIV who are combination antiretroviral therapy adherent, virally suppressed, and self-report cognitive symptoms.MethodsMRI, blood draws, and standardized neuropsychological test scores were collected from HIV-infected individuals. DTI metrics (fractional anisotropy, mean diffusivity, radial diffusivity, axial diffusivity) and plasma biomarkers (soluble CD163, soluble CD14, neopterin, IFN γ-induced protein 10, monocyte chemoattractant protein 1) were quantified. Statistical analysis explored associations between biomarker levels or neuropsychological test scores and DTI metrics using region of interest analyses and a voxelwise approach.ResultsA total of 43 participants with median (interquartile range) age of 64 (62-66 years), CD4 cell count of 600 (400-760 cell/μl) who were all virally suppressed (

The prevalence of age-related comorbidities is increased in people living with HIV, even in those well-controlled on combination antiretroviral therapy (ART). Persistent immune activation and inflammation may play pivotal roles in the pathogenesis; however, the burden of morbidities in the older HIV infected population may be exacerbated and driven by distinct mechanisms. In a cross sectional study of 45 HIV-infected participants 60 years or older, we examined the relationships between 14 immunomodulatory and inflammatory factors and the Veterans Aging Cohort Study (VACS) Index, a metric of multimorbidity and mortality comprised of age, CD4 count, hemoglobin, Fibrosis-4 [FIB-4], and estimated glomerular filtration rate [eGFR], by linear regression analysis. All participants were virally suppressed (

ObjectivesNearly half of the population living with human immunodeficiency virus (HIV) in the United States is now older than 50 years with at least 6% over age 65. Between 35% and 50% live with mild to moderate cognitive impairment. Older persons living with HIV (PLWH) also have a substantial burden of HIV-associated non-acquired immunodeficiency syndrome medical conditions and are at risk for frailty, geriatric syndromes, and early mortality compared with HIV-uninfected peers. We sought to define the magnitude of geriatric conditions and multimorbidity in PLWH older than 60 years who are living with symptomatic cognitive impairment. In a subset of participants, we examined associations between these geriatric conditions.DesignRetrospective cohort study.SettingHIV Elders Study at the University of California, San Francisco, Memory and Aging Center.ParticipantsParticipants were HIV infected, virally suppressed, 60 years or older, and clinically diagnosed with mild neurocognitive disorder (MND).MeasurementsWe conducted standardized assessment of geriatric conditions and everyday function and investigated multimorbidity burden using the Veterans Aging Cohort Study (VACS) index.ResultsAmong 141 older PLWH with MND, 58% report incontinence, 55% meet criteria for pre-frailty, and a substantial proportion report dependence with instrumental activities of daily living (52%) or activities of daily living (41%). The mean VACS index score is 33 (standard deviation = 14), suggesting a 13.8% 5-year all-cause mortality risk.ConclusionsOlder PLWH with symptomatic cognitive impairment carry a substantial burden of other geriatric conditions. Our work supports the need for comprehensive geriatric systems of care for cognitively impaired individuals aging with HIV. J Am Geriatr Soc 67:1913-1916, 2019.

We previously reported that galectin-9 (Gal-9), a soluble lectin with immunomodulatory properties, is elevated in plasma during HIV infection and induces HIV transcription. The link between Gal-9 and compromised neuronal function is becoming increasingly evident; however, the association with neuroHIV remains unknown. We measured Gal-9 levels by ELISA in cerebrospinal fluid (CSF) and plasma of 70 HIV-infected (HIV+) adults stratified by age (older > 40 years and younger

Human T-cell leukemia virus type 1 (HTLV-1) has infected as many as 10 million people worldwide. While 90% are asymptomatic, 5% develop severe diseases including adult T-cell leukemia/lymphoka (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). No vaccine against HTLV-1 exists, and screening programs are not universal. However, patients with chronic HTLV-1 infection have high frequencies of HTLV-1-activated CD8+ T cells, and the two main HLA alleles (A2, A24) are present in 88% of infected individuals. We thus utilized an immunoproteomics approach to characterize MHC-I restricted epitopes presented by HLA-A2+, A24+ MT-2 and SLB-1 cell lines. Unlike traditional motif prediction algorithms, this approach identifies epitopes associated with cytotoxic T-cell responses in their naturally processed forms, minimizing differences in antigen processing and protein expression levels. Out of nine identified peptides, we confirmed six novel MHC-I restricted epitopes that were capable of binding HLA-A2 and HLA-A24 alleles and used in vitro and in vivo methods to generate CD8+ T cells specific for each of these peptides. MagPix MILLIPLEX data showed that in vitro generated epitope-specific CD8+ T cells secreted IFN-ɣ, granzyme B, MIP-1α, TNF-α, perforin and IL-10 when cultured in the presence of MT-2 cell line. Degranulation assay confirmed cytotoxic response through surface expression of CD107 on CD8+ T cells when cultured with MT-2 cells. A CD8+ T-cell killing assay indicated significant antiviral activity of CD8+ T cells specific against all identified peptides. In vivo generated CD8+ T cells similarly demonstrated immunogenicity on ELISpot, CD107 degranulation assay, and MagPix MILLIPLEX analysis. These epitopes are thus candidates for a therapeutic peptide-based vaccine against HTLV-1, and our results provide preclinical data for the advancement of such a vaccine.

Plasminogen activator inhibitor type 1 (PAI-1), a key negative regulator of fibrinolysis, has been investigated to be one of the potential mechanisms of the development of impaired insulin sensitivity, insulin resistance, and diabetes mellitus. Because chronically stable HIV-infected individuals frequently develop abnormal glucose metabolism, including insulin resistance and diabetes mellitus, we postulated that PAI-1 could be one of the multifactorial pathogenic roles in the development of impaired insulin sensitivity and insulin resistance among chronic HIV-infected individuals. From our longitudinal cohort study, we selectively recruited chronically stable HIV-infected individuals without diagnosis of diabetes mellitus at baseline (N = 62) to analyze the correlation of baseline inflammatory cytokines, including PAI-1 and whole-body insulin sensitivity, with 2-year follow-up, as measured by Matsuda Index. We found a negative correlation between baseline PAI-1 and Matsuda Index (r = -0.435, p = .001) and a negative correlation between baseline PAI-1 and Matsuda Index at 2 years (r = -0.377, p = .005). In a linear regression model that included age, total body fat mass percentage, serum amyloid A, and family history of diabetes mellitus, PAI-1 still remained significantly associated with Matsuda Index at 2-year follow-up (β = -.397, p = .002). Our longitudinal study suggests that PAI-1 is an independent predictor of impaired insulin sensitivity among chronic HIV-infected individuals.

Inflammation associated with low 25-hydroxyvitamin D (25(OH)D) is associated with increased morbidity and mortality among HIV-infected patients with vitamin D deficiency. We investigated the association between 25(OH)D and soluble biomarkers among HIV-infected patients on stable antiretroviral therapy. This is a cross-sectional study. This study focuses on assessment in subjects 40 years or older on stable antiretroviral therapy (ART) for >3 months. Chemiluminescent immunoassay was used to determine plasma 25(OH)D levels. Plasma soluble biomarkers were measured by Luminex technology. Multivariable linear regression analysis was used to assess the associations between log10-25(OH)D and soluble biomarkers.Of 138 patients, median age was 50.5 (45, 57) years and 25(OH)D was 34.0 (25.0, 42.3) ng/mL. The majority were males (88%) and had undetectable HIV RNA (84.8%); 19 (13.8%) had 25(OH)D ≥50 ng/mL. Spline regression analyses suggested a J-shaped relationship between various plasma biomarkers and 25(OH)D. Among subjects with 25(OH)D ≥20 ng/mL, multivariable linear regression showed positive association between 25(OH)D and interleukin (IL)-10 (β = 1.84, P

Two innovative studies recently identified functional lymphatic structures in the meninges that may influence the development of HIV-associated neurological disorders (HAND). Until now, blood vessels were assumed to be the sole transport system by which HIV-infected monocytes entered the brain by bypassing a potentially hostile blood-brain barrier through inflammatory-mediated semi-permeability. A cascade of specific chemokine signals promote monocyte migration from blood vessels to surrounding brain tissues via a well-supported endothelium, where the cells differentiate into tissue macrophages capable of productive HIV infection. Lymphatic vessels on the other hand are more loosely organized than blood vessels. They absorb interstitial fluid from bodily tissues where HIV may persist and exchange a variety of immune cells (CD4(+) T cells, monocytes, macrophages, and dendritic cells) with surrounding tissues through discontinuous endothelial junctions. We propose that the newly discovered meningeal lymphatics are key to HIV migration among viral reservoirs and brain tissue during periods of undetectable plasma viral loads due to suppressive combinational antiretroviral therapy, thus redefining the migration process in terms of a blood-lymphatic transport system.

BackgroundInflammation may contribute to cardiovascular disease (CVD) among antiretrovirally suppressed HIV-infected individuals. We assessed relationships of monocyte, CD8 T-cell activation and plasma biomarkers to changes in carotid artery intima-media thickness (CIMT).MethodsLongitudinal study of HIV-infected subjects ≥40 years and on stable antiretroviral therapy (ART) ≥3 months. Peripheral blood mononuclear cells were immunophenotyped by multiparameteric flow cytometry to quantify classical (CD14(++)CD16(-)), intermediate (CD14(++)CD16(+)), non-classical (CD14(low/+)CD16(++)) and transitional (CD14(+)CD16(-)) monocyte subsets and activated (CD38(+)HLA-DR(+)) CD8(+) T-cells at baseline. Plasma biomarkers were assessed by multiplex Luminex assay. High-resolution B-mode ultrasounds of right carotid arteries were obtained. Changes in CIMT over two years at the right common carotid artery (CIMTCCA) and right bifurcation (CIMTBIF) were outcome variables.ResultsWe studied 50 subjects: 84% male, median age 49 (Q1, Q3; 46, 56) years, median CD4 count 461 (317, 578) cells/mm(3), and with HIV RNA ≤ 50 copies/mL in 84%. Change in CIMTBIF correlated with log values of baseline absolute count of non-classical monocytes (r = 0.37, p = 0.020), and with MCP-1 (r = 0.42, p = 0.0024) and TNF-α (r = 0.30, p = 0.036) levels. In multivariable linear regression, only non-classical monocytes and MCP-1 predicted the change in CIMTBIF, independent of Framingham Risk Score and baseline CIMTBIF. No correlation was noted between CD8 T-cell activation and CIMTBIF change. Monocyte subsets, CD8 T-cell activation, and biomarker concentrations were not correlated with changes in CIMTCCA.ConclusionsOur findings highlight the role of non-classical monocytes and MCP-1 in the progression of CIMTBIF in HIV-infected individuals on stable ART independent of traditional cardio-metabolic risk factors.

Identifying host immune determinants governing HIV transcription, latency and infectivity in vivo is critical to developing an HIV cure. Based on our recent finding that the host factor p21 regulates HIV transcription during antiretroviral therapy (ART), and published data demonstrating that the human carbohydrate-binding immunomodulatory protein galectin-9 regulates p21, we hypothesized that galectin-9 modulates HIV transcription. We report that the administration of a recombinant, stable form of galectin-9 (rGal-9) potently reverses HIV latency in vitro in the J-Lat HIV latency model. Furthermore, rGal-9 reverses HIV latency ex vivo in primary CD4+ T cells from HIV-infected, ART-suppressed individuals (p = 0.002), more potently than vorinostat (p = 0.02). rGal-9 co-administration with the latency reversal agent "JQ1", a bromodomain inhibitor, exhibits synergistic activity (p

HIV infection induces phenotypic and functional changes to CD8+ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8+ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT+ and TIGIT+ PD-1+ CD8+ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8+ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8+ T cell effector responses. The frequency of TIGIT+ CD4+ T cells correlated with the CD4+ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.

UnlabelledChronic HIV infection results in a loss of HIV-specific CD8(+) T cell effector function, termed "exhaustion," which is mediated, in part, by the membrane coinhibitory receptor T cell immunoglobulin mucin domain-3 (Tim-3). Like many other receptors, a soluble form of this protein has been described in human blood plasma. However, soluble Tim-3 (sTim-3) is poorly characterized, and its role in HIV disease is unknown. Here, we show that Tim-3 is shed from the surface of responding CD8(+) T cells by the matrix metalloproteinase ADAM10, producing a soluble form of the coinhibitory receptor. Despite previous reports in the mouse model, no alternatively spliced, soluble form of Tim-3 was observed in humans. Shed sTim-3 was found in human plasma and was significantly elevated during early and chronic untreated HIV infection, but it was not found differentially modulated in highly active antiretroviral therapy (HAART)-treated HIV-infected subjects or in elite controllers compared to HIV-uninfected subjects. Plasma sTim-3 levels were positively correlated with HIV load and negatively correlated with CD4 counts. Thus, plasma sTim-3 shedding correlated with HIV disease progression. Despite these correlations, we found that shedding Tim-3 did not improve the function of CD8(+) T cells in terms of gamma interferon production or prevent their apoptosis through galectin-9. Further characterization studies of sTim-3 function are needed to understand the contribution of sTim-3 in HIV disease pathogenesis, with implications for novel therapeutic interventions.ImportanceDespite the overall success of HAART in slowing the progression to AIDS in HIV-infected subjects, chronic immune activation and T cell exhaustion contribute to the eventual deterioration of the immune system. Understanding these processes will aid in the development of interventions and therapeutics to be used in combination with HAART to slow or reverse this deterioration. Here, we show that a soluble form of T cell exhaustion associated coinhibitory molecule 3, sTim-3, is shed from the surface of T cells. Furthermore, sTim-3 is elevated in the plasma of treatment-naive subjects with acute or chronic HIV infection and is associated with markers of disease progression. This is the first study to characterize sTim-3 in human plasma, its source, and mechanism of production. While it is still unclear whether sTim-3 contributes to HIV pathogenesis, sTim-3 may represent a new correlate of HIV disease progression.

The T cell Ig- and mucin domain-containing molecule-3 (Tim-3) negative immune checkpoint receptor demarcates functionally exhausted CD8(+) T cells arising from chronic stimulation in viral infections like HIV. Tim-3 blockade leads to improved antiviral CD8(+) T cell responses in vitro and, therefore, represents a novel intervention strategy to restore T cell function in vivo and protect from disease progression. However, the Tim-3 pathway in the physiologically relevant rhesus macaque SIV model of AIDS remains uncharacterized. We report that Tim-3(+)CD8(+) T cell frequencies are significantly increased in lymph nodes, but not in peripheral blood, in SIV-infected animals. Tim-3(+)PD-1(+)CD8(+) T cells are similarly increased during SIV infection and positively correlate with SIV plasma viremia. Tim-3 expression was found primarily on effector memory CD8(+) T cells in all tissues examined. Tim-3(+)CD8(+) T cells have lower Ki-67 content and minimal cytokine responses to SIV compared with Tim-3(-)CD8(+) T cells. During acute-phase SIV replication, Tim-3 expression peaked on SIV-specific CD8(+) T cells by 2 wk postinfection and then rapidly diminished, irrespective of mutational escape of cognate Ag, suggesting non-TCR-driven mechanisms for Tim-3 expression. Thus, rhesus Tim-3 in SIV infection partially mimics human Tim-3 in HIV infection and may serve as a novel model for targeted studies focused on rejuvenating HIV-specific CD8(+) T cell responses.

Galectin-9 (Gal-9) is a β-galactosidase-binding lectin that promotes apoptosis, tissue inflammation, and T cell immune exhaustion, and alters HIV infection in part through engagement with the T cell immunoglobulin mucin domain-3 (Tim-3) receptor and protein disulfide isomerases (PDI). Gal-9 was initially thought to be an eosinophil attractant, but is now known to mediate multiple complex signaling events that affect T cells in both an immunosuppressive and inflammatory manner. To understand the kinetics of circulating Gal-9 levels during HIV infection we measured Gal-9 in plasma during HIV acquisition, in subjects with chronic HIV infection with differing virus control, and in uninfected individuals. During acute HIV infection, circulating Gal-9 was detected as early as 5 days after quantifiable HIV RNA and tracked plasma levels of interleukin (IL)-10, tumor necrosis factor (TNF)-α, and IL-1β. In chronic HIV infection, Gal-9 levels positively correlated with plasma HIV RNA levels (r=0.29; p=0.023), and remained significantly elevated during suppressive antiretroviral therapy (median: 225.3 pg/ml) and in elite controllers (263.3 pg/ml) compared to age-matched HIV-uninfected controls (54 pg/ml). Our findings identify Gal-9 as a novel component of the first wave of the cytokine storm in acute HIV infection that is sustained at elevated levels in virally suppressed subjects and suggest that Gal-9:Tim-3 crosstalk remains active in elite controllers and antiretroviral (ARV)-suppressed subjects, potentially contributing to ongoing inflammation and persistent T cell dysfunction.

Coronary artery calcium (CAC) is a validated subclinical measure of atherosclerosis. Studies in the general population have linked blood inflammatory biomarkers including monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor (TNF)-α with the burden of CAC, but this relationship is often lost following correction for traditional cardiovascular risk factors. We assessed the relationship of various biomarkers to CAC, specifically in HIV-infected individuals on potent antiretroviral therapy (ART). Analyses utilized entry data from participants in the Hawaii Aging with HIV-Cardiovascular (HAHC-CVD) study. Computerized tomography examinations for CAC were obtained locally and analyzed by a central reading center in blinded fashion. Plasma biomarkers were assessed by multiplexing using Milliplex Human Cardiovascular Disease panels. Among a cohort of 130 subjects [88% male, median (IQR) age of 51 (46-57) years, CD4 count of 492 (341-635) cells/mm(3), 86.9% with HIV RNA ≤50 copies/ml], CAC was present in 46.9% of subjects. In univariate analyses higher levels of log-transformed MCP-1 and TNF-α were associated with the presence of CAC (p

Little is known about the manipulation of IL-17 producing CD4+ T cells (TH17) on a per-cell basis in humans in vivo. Previous studies on the effects of IL-2 on IL-17 secretion in non-HIV models have shown divergent results. We hypothesized that IL-2 would mediate changes in IL-17 levels among recently HIV-1-infected adults receiving anti-retroviral therapy. We measured cytokine T cell responses to CD3/CD28, HIV-1 Gag, and CMV pp65 stimulation, and changes in multiple CD4+ T cell subsets. Those who received IL-2 showed a robust expansion of naive and total CD4+ T cell counts and T-reg counts. However, after IL-2 treatment, the frequency of TH17 cells declined, while counts of TH17 cells did not change due to an expansion of the CD4+ naïve T cell population (CD27+CD45RA+). Counts of HIV-1 Gag-specific T cells declined modestly, but CMV pp65 and CD3/CD28 stimulated populations did not change. Hence, in contrast with recent studies, our results suggest IL-2 is not a potent in vivo regulator of TH17 cell populations in HIV-1 disease. However, IL-2-mediated T-reg expansions may selectively reduce responses to certain antigen-specific populations, such as HIV-1 Gag.

BackgroundThe relationship of elevated T cell activation to altered T cell differentiation profiles, each defining features of HIV-1 infection, has not been extensively explored. We hypothesized that anti-retroviral suppression of T cell activation levels would lead to alterations in the T cell differentiation of total and HIV-1 specific CD8+ T cell responses among recently HIV-1 infected adults.Methodology/principal findingsWe performed a longitudinal study simultaneously measuring T cell activation and maturation markers on both total and antigen-specific T cells in recently infected adults: prior to treatment; after the initiation of HAART; and after treatment was halted. Prior to treatment, HIV-1 Gag-specific CD8+ T cells were predominantly of a highly activated, intermediate memory (CD27+CD28-) phenotype, while CMV pp65-specific CD8+ T cells showed a late memory (CD27-CD28-), low activation phenotype. Participants with the highest fraction of late memory (CD27-CD28-) HIV-1-specific CD8+ T cells had higher CD4+ T cell counts (rho = +0.74, p = 0.004). In turn, those with the highest fraction of intermediate memory (CD27+ CD28-) HIV-1 specific CD8+ T cells had high total CD8+ T cell activation (rho = +0.68, p = 0.01), indicating poorer long-term clinical outcomes. The HIV-1 specific T cell differentiation profile was not readily altered by suppression of T cell activation following HAART treatment.Conclusions/significanceA more differentiated, less activated HIV-1 specific CD8+ T cell response may be clinically protective. Anti-retroviral treatment initiated two to four months after infection lowered T cell activation but had no effect on the differentiation profile of the HIV-1-specific response. Intervention during the first month of acute infection may be required to shift the differentiation phenotype of HIV-1 specific responses to a more clinically favorable profile.

Human endogenous retroviruses (HERVs) are remnants of ancient infectious agents that have integrated into the human genome. Under normal circumstances, HERVs are functionally defective or controlled by host factors. In HIV-1-infected individuals, intracellular defense mechanisms are compromised. We hypothesized that HIV-1 infection would remove or alter controls on HERV activity. Expression of HERV could potentially stimulate a T cell response to HERV antigens, and in regions of HIV-1/HERV similarity, these T cells could be cross-reactive. We determined that the levels of HERV production in HIV-1-positive individuals exceed those of HIV-1-negative controls. To investigate the impact of HERV activity on specific immunity, we examined T cell responses to HERV peptides in 29 HIV-1-positive and 13 HIV-1-negative study participants. We report T cell responses to peptides derived from regions of HERV detected by ELISPOT analysis in the HIV-1-positive study participants. We show an inverse correlation between anti-HERV T cell responses and HIV-1 plasma viral load. In HIV-1-positive individuals, we demonstrate that HERV-specific T cells are capable of killing cells presenting their cognate peptide. These data indicate that HIV-1 infection leads to HERV expression and stimulation of a HERV-specific CD8+ T cell response. HERV-specific CD8+ T cells have characteristics consistent with an important role in the response to HIV-1 infection: a phenotype similar to that of T cells responding to an effectively controlled virus (cytomegalovirus), an inverse correlation with HIV-1 plasma viral load, and the ability to lyse cells presenting their target peptide. These characteristics suggest that elicitation of anti-HERV-specific immune responses is a novel approach to immunotherapeutic vaccination. As endogenous retroviral sequences are fixed in the human genome, they provide a stable target, and HERV-specific T cells could recognize a cell infected by any HIV-1 viral variant. HERV-specific immunity is an important new avenue for investigation in HIV-1 pathogenesis and vaccine design.

Objective: High rates of albuminuria are observed among HIV-infected individuals on stable antiretroviral therapy (ART). Though pro-inflammatory and pro-fibrotic responses are described as components of albuminuria in the general population, it is unclear how these responses are associated to albuminuria in ART-treated chronic HIV. We investigated the relationship of monocyte subsets and urine inflammatory and fibrotic biomarkers to albuminuria in ART-treated HIV-infected participants. Design and Methods: Cross-sectional analyses were performed on Hawaii Aging with HIV-cardiovascular disease study cohort participants who were required at entry to be ≥40 years old and on ART ≥3 months. Monocyte subpopulations were determined in banked peripheral blood mononuclear cells (PBMC) using multi-parametric flow-cytometry. Entry random urine samples were assessed for albumin-to-creatinine ratios (UACR). Urine samples were measured for inflammatory and fibrotic biomarkers using Luminex technology. Results: Among 96 HIV-infected subjects with measured UACR (87% male, 59% Caucasian, and 89% undetectable HIV RNA with median CD4 of 495.5 cells/μL), 18 patients (19%) had albuminuria. Non-classical (CD14low/+CD16++) monocytes were significantly elevated in subjects with albuminuria (p = 0.034) and were correlated to UACR (r = 0.238, p = 0.019). Elevated non-classical monocyte counts were significant predictors of worsening albuminuria, independent of traditional- and ART-associated risk factors (β = 0.539, p = 0.007). Urine TGF-β1 and collagen-IV were significantly higher in albuminuric compared to non-albuminuric participants (TGF-β1; p = 0.039 and collagen-IV; p = 0.042). Urine TGF-β1 was significantly correlated with non-classical monocyte counts (r = 0.464, p = 0.017). Conclusion: Alterations in monocyte subpopulations and urine pro-fibrotic factors may play a role in kidney dysfunction during chronic HIV infection and warrants further study. [ABSTRACT FROM AUTHOR]

Background: Persistent inflammation and immune activation has been hypothesized to contribute to increased prevalence of subclinical atherosclerosis and cardiovascular disease (CVD) risk in patients with chronic HIV infection. In this study, we examined the correlation of peripheral monocyte subsets and soluble biomarkers of inflammation to coronary artery calcium (CAC) progression, as measured by cardiac computed tomography scan. Methods: We conducted a longitudinal analysis utilizing baseline data of 78 participants with HIV infection on stable antiretroviral therapy (ART) in the Hawaii Aging with HIV-Cardiovascular study who had available baseline monocyte subset analysis as well as CAC measurement at baseline and at 2-year follow up. Monocyte phenotypes were assessed from cryopreserved blood by flow cytometry and plasma was assayed for soluble biomarkers using antibody-coated beads in a high sensitivity Milliplex Luminex platform. Change in CAC over 2 years was analyzed as the primary outcome variable. Results: Of all monocyte subsets and biomarkers tested, higher non-classical monocyte percentage (ρ = 0.259, p = 0.022), interleukin (IL)-6 (ρ = 0.311, p = 0.012), and monocyte chemoattractant protein (MCP)-1 (ρ = 0.524, p = <0.001) were significantly correlated to higher 2-year CAC progression in unadjusted Spearman’s correlation. Non-classical monocyte percentage (ρ = 0.247, p = 0.039), and MCP-1 (ρ = 0.487, p = <0.001), remained significantly correlated to 2-year CAC progression, while IL-6 was not (ρ = 0.209, p = 0.120) after adjustment for age, hypertension, diabetes mellitus, total/HDL cholesterol ratio, smoking history, and BMI. Conclusion: The percentage of non-classical monocytes and plasma MCP-1 levels were independently associated with CAC progression and may be related to the progression of atherosclerosis and increased CVD risk associated with chronic HIV infection on stable ART. [ABSTRACT FROM AUTHOR]

More than a decade after West Nile virus (WNV) entered North America, and despite a significant increase in reported cases during the 2012 and 2013 seasons, no treatment or vaccine for humans is available. Although antiviral T cells contribute to the control of WNV, little is known about their regulation during acute infection. We analyzed the expression of Tim-3 and PD-1, two recently identified T cell negative immune checkpoint receptors, over the course of WNV infection. Symptomatic WNV+ donors exhibited higher frequencies of Tim-3+ cells than asymptomatic subjects within naïve/early differentiated CD28+/–CD57–CD4+ and differentiated CD28–CD57–CD8+ T cells. Our study links Tim-3-expression on T cells during acute WNV infection with the development of symptomatic disease, suggesting Tim-3 and its ligands could be targeted therapeutically to alter anti-WNV immunity and improve disease outcome. [ABSTRACT FROM AUTHOR]

Background: Rates of insulin resistance are increased in HIV-infected patients on stable antiretroviral therapy (ART). Such increase may partially be due to HIV-induced immune dysregulation involving monocytes (MO) and its subsets. Materials and Methods: Cross-sectional analysis of 141 HIV-infected subjects age ≥ 40 years on stable ART. Homeostatic model assessment–insulin resistance (HOMA-IR) and rates of metabolic syndrome were calculated. Subjects were classified by fasting glucose and oral glucose tolerance test (OGTT) into clinical diabetes categories. Multi-parametric flow cytometry was used to determine MO subset percentages: [classical (CD14++CD16−), intermediate (CD14++CD16+), non-classical (CD14low/+CD16++), and a recently identified fourth (CD14low/+CD16−) ‘transitional’ MO subset] and percentage of activated (CD38+HLA-DR+) CD8 T cells. Absolute levels of cells were calculated using clinical CBC and T cell subset data. Multiple plasma soluble biomarkers were assessed by Luminex technology. Results: Median age 50 years, CD4 count (percent) 505 cells/µL (29%), and 89% male. Total MO (r = −0.23, p = 0.006) and classical and non-classical MO subsets correlated negatively with CD4 percent. No correlations were seen with CD4 count as absolute values. Log-total MO and log-classical MO predicted HOMA-IR independently of HIV immuno-virologic and diabetes risk factors (β = 0.42, p = 0.02 and β = 0.35, p = 0.02, respectively) and were increased in subjects with metabolic syndrome (p = 0.03 and p = 0.05 respectively). Total and/or subset MO levels correlated with multiple soluble plasma biomarkers including CRP, IL-6, MMP-9, MPO, SAA, SAP and tPAI-1, with tPAI-1 independently predicting HOMA-IR (β = 0.74, p<0.001). Conclusions: MO levels increase with worsening HIV immune dysregulation as assessed by CD4 percent. CD4 percent may provide additional information about MO and metabolic risk in this population beyond absolute values. MO, and specifically classical MO, may contribute to insulin resistance and metabolic syndrome during chronic HIV infection. Multiple soluble plasma biomarkers including tPAI-1 increase with increase in MO. Levels of tPAI-1 independently predict the development of insulin resistance. [ABSTRACT FROM AUTHOR]