Human Ag-specific CD4 + T cells can be detected by their dual expression of CD134 (OX40) and CD25 after a 44 hours stimulation with cognate Ag. We show that surface expression of CD39 on Ag-specific cells consistently identifies a substantial population of CD4 + CD25 + CD134 + CD39 + T cells that have a Treg-cell-like phenotype and mostly originate from bulk memory CD4 + CD45RO + CD127 low CD25 high CD39 + Treg cells. Viable, Ag-specific CD25 + CD134 + CD39 + T cells could be expanded in vitro as cell lines and clones, and retained high Forkhead Box Protein 3, CTLA-4 and CD39 expression, suppressive activity and Ag specificity. We also utilised this combination of cell surface markers to measure HIV-Gag responses in HIV + patients before and after anti-retroviral therapy (ART). Interestingly, we found that the percentage of CD39 − cells within baseline CD4 + T-cell responses to HIV-Gag was negatively correlated with HIV viral load pre-ART and positively correlated with CD4 + T-cell recovery over 96 weeks of ART. Collectively, our data show that Ag-specific CD4 + CD25 + CD134 + CD39 + T cells are highly enriched for Treg cells, form a large component of recall responses and maintain a Treg-cell-like phenotype upon in vitro expansion. Identification and isolation of these cells enables the role of Treg cells in memory responses to be further defined and provides a development pathway for novel therapeutics.Keywords: Antigen-specific T cells r CD25 r CD39 r CD134 r FOXP3 r OX40 r Treg cell Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Laura Cook e-mail: lcook@kirby.unsw.edu.au * These authors contributed equally to this work. The current gold standard marker for the study of Treg cells is Forkhead Box Protein 3 (FOXP3), a constitutively expressed nuclear transcription factor, critical for Treg-cell survival and suppressive function [3]. The key limitation of this marker is that viable cells cannot be isolated, as staining for this intracellular protein involves cell permeabilisation. Also, in the context of activation, this marker loses specificity due to transient, activationinduced up-regulation of FOXP3 in human non-Treg cells [4,5]. An alternative, widely used definition of Treg cells is the combined cell surface expression of high levels of CD25 (IL-2Rα) and low levels of CD127 (IL-7Rα) [6]. Whilst >85% of CD127 low CD25 high Treg cells express FOXP3 and are suppressive ex vivo [6], activated Treg cells cannot be isolated with these markers due to CD127 down-regulation, and CD25 up-regulation, on other subsets of CD4 + T cells following activation by cognate .In 2007, it was shown that murine Treg cells co-express the ectoenzymes CD39 and CD73 [10]. These ectonucleotidases work in concert to convert adenosine triphosphate (ATP), adenosine diphosphate and adenosine monophosphate to immunosuppressive adenosine, which appears to play a role in the suppressive repertoire of Treg cells [10]. Apart from contributing to adenosine produ...

Objective To determine the associations of markers of immune activation with atherosclerosis and mortality, in participants with treated and suppressed HIV infection. Design Observational study of 149 HIV-infected participants with virologic suppression on antiretroviral therapy. Methods Cryopreserved mononuclear cells and plasma were used to evaluate markers of T cell and monocyte activation, inflammation and coagulopathy. Carotid artery intima-media thickness (CIMT) was measured by high-resolution ultrasound at the common, bifurcation and internal carotid regions. Associations of immunologic markers with CIMT and all-cause mortality were assessed using multivariable linear regression and Cox proportional hazards regression. Results The majority of participants were male (93%) and white (67%), median age of 48.5 years and median CD4+ T cell count of 522 cells/μL. The median baseline IMT was 1.0 mm. Over a median of 8.3 years of follow-up, 12 deaths occurred. In multivariate analysis, adjusted for traditional cardiovascular risk factors, higher monocyte CCR5 expression (5.4%, 95%CI [2.4–8.4], p=0.001) was associated with greater common carotid IMT. Higher plasma IL-6 was associated with greater bifurcation (8.0%, 95%CI [2.3–13.7], p=0.007) and overall mean IMT (5.2%, 95%CI [0.7–9.7], p=0.026). Finally, higher plasma IL-6 (HR 1.9, 95%CI [1.0–3.7], p=0.030), internal carotid (HR 4.1, 95%CI [1.2–13.7], p=0.022) and mean IMT (HR 5.2, 95%CI [1.2–22.1], p=0.026) were individually associated with all-cause mortality. Conclusions Higher monocyte CCR5 expression and plasma IL-6 were associated with atherosclerosis, independent of traditional cardiovascular risk factors. IL-6 and CIMT were individually associated with all-cause mortality. The impact of therapies targeting immune activation in CVD in treated HIV infection merits additional investigation.

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Background HIV infection is associated with a high risk of cardiovascular diseases (CVD) and increased arterial inflammation. In HIV, inflammation is also increased within lymph nodes (LN), tissues known to harbor the virus even among treated and suppressed individuals. We tested the hypothesis that arterial inflammation is linked to HIV disease activity and to inflammation within HIV-infected tissues (LN). Methods Seventy-four individuals were studied: 45 HIV-infected individuals and 29 uninfected controls. Arterial and LN inflammation were measured using 18F-fluorodeoxyglucose positron emission tomography, (FDG-PET). Detailed immunophenotyping was performed, along with measurement of viral activity/persistence, and circulating inflammatory biomarkers. Results Median age was 53 years, 100% male. Lymph node inflammation was higher in HIV-infected individuals and correlated with markers of viral disease activity (viral load, CD8+ T cells, CD4/CD8 ratio) and CD4+ T cell activation. Arterial inflammation was modestly increased in HIV-infected individuals and was positively correlated with circulating inflammatory biomarkers (hsCRP, IL-6) and activated monocytes (CD14dimCD16+; non-classical) but not to markers of HIV. While LN and arterial inflammation were increased in HIV, inflammatory activity in these tissues was not related (r=0.09, p=0.56). Conclusion While LN and to a lesser degree, the arterial wall are inflamed in HIV, inflammation in these tissues is not closely linked. Namely, measures of HIV disease activity strongly associate with LN inflammation but not arterial inflammation. These data suggest that LN and arterial inflammation do not share underlying pathways of immune activation and suggests therapeutic interventions that reduce viral disease activity may not predictably reduce arterial inflammation in HIV or its down-stream consequence (CVD).

Despite the major advances in the management of HIV infection, HIV-infected patients still have greater morbidity and mortality than the general population. Serious non-AIDS events (SNAEs), including non-AIDS malignancies, cardiovascular events, renal and hepatic disease, bone disorders and neurocognitive impairment, have become the major causes of morbidity and mortality in the antiretroviral therapy (ART) era. SNAEs occur at the rate of 1 to 2 per 100 person-years of follow-up. The pathogenesis of SNAEs is multifactorial and includes the direct effect of HIV and associated immunodeficiency, underlying co-infections and co-morbidities, immune activation with associated inflammation and coagulopathy as well as ART toxicities. A number of novel strategies such as ART intensification, treatment of co-infection, the use of anti-inflammatory drugs and agents that reduce microbial translocation are currently being examined for their potential effects in reducing immune activation and SNAEs. However, currently, initiation of ART before advanced immunodeficiency, smoking cessation, optimisation of cardiovascular risk factors and treatment of HCV infection are most strongly linked with reduced risk of SNAEs or mortality. Clinicians should therefore focus their attention on addressing these issues prior to the availability of further data.

In the ART era, serious non-AIDS events (SNAEs) have become the major causes of morbidity and mortality in HIV-infected persons. Early ART initiation has the strongest evidence for reducing SNAEs and mortality. Biomarkers of immune activation, inflammation and coagulopathy do not fully normalize despite virologic suppression and persistent immune activation is an important contributor to SNAEs. A number of strategies aimed to reduce persistent immune activation including ART intensification to reduce residual viremia; treatment of co-infections to reduce chronic antigen stimulation; the use of anti-inflammatory agents, reducing microbial translocation as well as interventions to improve immune recovery through cytokine administration and reducing lymphoid tissue fibrosis have been investigated. To date, there is little conclusive evidence on which strategies beyond treatment of hepatitis B and C co-infections and reducing cardiovascular risk factors will result in clinical benefits in patients already on ART with viral suppression. The use of statins seems to show early promise and larger clinical trials are underway to confirm their efficacy. At this stage, clinical care of HIV-infected patients should therefore focus on early diagnosis and prompt ART initiation, treatment of active co-infections and the aggressive management of co-morbidities until further data are available.

Studies utilizing highly pathogenic simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) have largely focused on the immunopathology of the central nervous system (CNS) during end-stage neurological AIDS and SIV encephalitis. However, this may not model pathophysiology in earlier stages of infection. In this nonaccelerated SHIV model, plasma SHIV RNA levels and peripheral blood and colonic CD4+ T cell counts mirrored early human immunodeficiency virus (HIV) infection in humans. At 12 weeks postinfection, cerebrospinal fluid (CSF) detection of SHIV RNA and elevations in IP-10 and MCP-1 reflected a discrete neurovirologic process. Immunohistochemical staining revealed a diffuse, low-level CD3+ CD4− cellular infiltrate in the brain parenchyma without a concomitant increase in CD68/CD163+ monocytes, macrophages, and activated microglial cells. Rare SHIV-infected cells in the brain parenchyma and meninges were identified by RNAScope in situ hybridization. In the meninges, there was also a trend toward increased CD4+ infiltration in SHIV-infected animals but no differences in CD68/CD163+ cells between SHIV-infected and uninfected control animals. These data suggest that in a model that closely recapitulates human disease, CNS inflammation and SHIV in CSF are predominantly mediated by T cell-mediated processes during early infection in both brain parenchyma and meninges. Because SHIV expresses an HIV rather than SIV envelope, this model could inform studies to understand potential HIV cure strategies targeting the HIV envelope.IMPORTANCE Animal models of the neurologic effects of HIV are needed because brain pathology is difficult to assess in humans. Many current models focus on the effects of late-stage disease utilizing SIV. In the era of antiretroviral therapy, manifestations of late-stage HIV are less common. Furthermore, new interventions, such as monoclonal antibodies and therapeutic vaccinations, target HIV envelope. We therefore describe a new model of central nervous system involvement in rhesus macaques infected with SHIV expressing HIV envelope in earlier, less aggressive stages of disease. Here, we demonstrate that SHIV mimics the early clinical course in humans and that early neurologic inflammation is characterized by predominantly T cell-mediated inflammation accompanied by SHIV infection in the brain and meninges. This model can be utilized to assess the effect of novel therapies targeted to HIV envelope on reducing brain inflammation before end-stage disease.

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