The Canadian Study of Arterial Inflammation in Patients with Diabetes and Recent Vascular Events, Evaluation of Colchicine Effectiveness (CADENCE): protocol for a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Inflammation is a key mediator in the development and progression of the atherosclerotic disease process as well as its resultant complications, like myocardial infarction (MI), stroke and cardiovascular (CV) death, and is emerging as a novel treatment target. Trials involving anti-inflammatory medications have demonstrated outcome benefit in patients with known CV disease. In this regard, colchicine appears to hold great promise. However, there are potential drawbacks to colchicine use, as some studies have identified an increased risk of infection, and a non-significant trend for increased all-cause mortality. Thus, a more thorough understanding of the underlying mechanism of action of colchicine is needed to enable a better patient selection for this novel CV therapy. OBJECTIVE: The primary objective of the Canadian Study of Arterial Inflammation in Patients with Diabetes and Recent Vascular Events, Evaluation of Colchicine Effectiveness (CADENCE) trial is to assess the effect of colchicine on vascular inflammation in the carotid arteries and ascending aorta measured with 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT in patients with type 2 diabetes mellitus (T2DM) or pre-diabetes who have experienced a recent vascular event (acute coronary syndrome (ACS)/MI, transient ischaemic attack (TIA) or stroke). Secondary objectives include determining colchicine's effect on inflammatory biomarkers (high-sensitivity C reactive protein (hs-CRP) and interleukin-6 (IL-6)). Additionally, we will assess if baseline inflammation imaging or biomarkers are associated with a treatment response to colchicine determined by imaging. Exploratory objectives will look at: (1) the difference in the inflammatory response to colchicine in patients with coronary events compared with patients with cerebral events; (2) the difference in the inflammatory response to colchicine in different vascular beds; (3) the relationship of FDG-PET imaging markers with serum biomarkers and (4) assessment of quality-of-life changes. METHODS AND DESIGN: CADENCE is a multicentre, prospective, randomised, double-blinded, placebo-controlled study to determine the effect of colchicine on arterial inflammation as assessed with imaging and circulatory biomarkers, specifically carotid arteries and aortic FDG uptake as well as hs-CRP and IL-6 among others. Patients with T2DM or pre-diabetes who have recently experienced a CV event (within 30-120 days after an ACS (ie, ST-elevation MI (STEMI) or non-STEMI)) or TIA/stroke with documented large vessel atherosclerotic disease will be randomised to treatment with either colchicine 0.6 mg oral daily or placebo. Participants will undergo baseline clinical evaluation including EQ5D assessment, blood work for inflammatory markers and FDG PET/CT scan of the ascending aorta and left and right carotid arteries. Patients will undergo treatment for 6 months and have repeat clinical evaluation including EQ5D assessment, blood work for inflammatory markers and FDG PET/CT scan at the conclusion of the study. The primary outcome will be the change in the maximum target to background ratio (TBRmax) in the ascending aorta (or carotid arteries) from baseline to follow-up on FDG PET/CT imaging. DISCUSSION: Colchicine is an exciting potential new therapy for CV risk reduction. However, its use is associated with side effects and greater understanding of its underlying mechanism of action is needed. Importantly, the current study will determine whether its anti-inflammatory action is an indirect systemic effect, or a more local plaque action that decreases inflammation. The results will also help identify patients who will benefit most from such therapy. TRIAL REGISTRATION NUMBER: NCT04181996.

"They're all struggling as well": social and economic barriers and facilitators to self-managing chronic illness among marginalized people who use drugs.

PURPOSE: Self-management is recommended for addressing chronic conditions, and self-management programmes improve health behaviours and outcomes. However, social and economic factors have been neglected in self-management research, despite their relevance for marginalized groups. Thus, we aimed to explore barriers and facilitators that influence self-management among socioeconomically marginalized people who use drugs (PWUD). METHODS: Using community-based participatory methods, we developed a qualitative interview guide and conducted peer-led recruitment. Participants were admitted into the study after self-identifying as using non-prescribed drugs, having a chronic health issue, and experiencing socioeconomic marginalization. Data were analysed using reflexive thematic analysis, taking a relational autonomy lens. RESULTS: Participants highlighted substantial barriers to managing their health issues, mostly stemming from their social and economic environments, such as unstable housing, low income, lack of supportive social networks, and negative healthcare experiences. Participants also described how their ability to self-manage their chronic conditions benefited from specific aspects of social interactions, including close relationships, community connectedness, and engaging in peer support. CONCLUSIONS: Our findings suggest that structural interventions are needed to support self-management among marginalized PWUD, especially stable housing. Self-management supports for PWUD would benefit from including a range of low-barrier community-based options, peer work opportunities, and advocacy for needs.

"The Drug Use Unfortunately isn't all Bad": Chronic Disease Self-Management Complexity and Strategy Among Marginalized People Who Use Drugs.

Self-management programs improve health outcomes and self-management is recommended for chronic conditions. Yet chronic disease self-management supports have rarely been applied to people who use drugs (PWUD). Thus, our objective was to explore self-management experiences among marginalized PWUD. We used community-based participatory methods and conducted qualitative interviews. Participants self-identified as having long-term and past year experience using non-prescribed drugs, one other chronic condition, and socioeconomic marginalization. We analyzed the data using reflexive thematic analysis. Although many participants considered drug use a chronic health issue, self-medicating with non-prescribed drugs was also a key self-management strategy to address other health issues. Participants also described numerous other strategies, including cognitive and behavioral tactics. These findings highlight the need for a safe supply of pharmaceutical-grade drugs to support self-management among marginalized PWUD. Self-management supports should also be tailored to address relevant topics (e.g., harm reduction, withdrawal), include creative activities, and not hinder PWUD's agency.

Routinized Syphilis Screening Among Men Living With Human Immunodeficiency Virus: A Stepped Wedge Cluster Randomized Controlled Trial.

BACKGROUND: We implemented an opt-out clinic-based intervention pairing syphilis tests with routine human immunodeficiency virus (HIV) viral load testing. The primary objective was to determine the degree to which this intervention increased the detection of early syphilis. METHODS: The Enhanced Syphilis Screening Among HIV-Positive Men (ESSAHM) Trial was a stepped wedge cluster-randomized controlled trial involving 4 urban HIV clinics in Ontario, Canada, from 2015 to 2017. The population was HIV-positive adult males. The intervention was standing orders for syphilis serological testing with viral loads, and control was usual practice. We obtained test results via linkage with the centralized provincial laboratory and defined cases using a standardized clinical worksheet and medical record review. We employed a generalized linear mixed model with a logit link to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of the intervention. RESULTS: A total of 3895 men were followed over 7471 person-years. The mean number of syphilis tests increased from 0.53 to 2.02 tests per person per year. There were 217 new diagnoses of syphilis (control, 81; intervention, 136), for which 147 (68%) were cases of early syphilis (control, 61 [75%]; intervention, 86 [63%]). The annualized proportion with newly detected early syphilis increased from 0.009 to 0.032 with implementation of the intervention; the corresponding time-adjusted OR was 1.25 (95% CI, .71-2.20). CONCLUSIONS: The implementation of standing orders for syphilis testing with HIV viral loads was feasible and increased testing, yet produced less-than-expected increases in case detection compared to past uncontrolled pre-post trials. CLINICAL TRIALS REGISTRATION: NCT02019043.

Psychological changes in successful completers of an HIV-tailored smoking cessation program: mood, attachment and self-efficacy.

High rates of cigarette smoking is the leading contributor to the increasing risk of cardiovascular disease (CVD) among people living with HIV (PLH). Relapse rates among PLH who quit smoking are high among those receiving standard care, which may be due to several unique social and psychological challenges PLH face when they attempt to quit smoking. The purpose of the current study was to examine change in relevant psychological factors in a subgroup of participants (n = 14) who remained smoke-free at 6-months follow-up in an HIV-tailored smoking cessation counselling program (N = 50). We examined self-reported depressive symptoms, attachment style and self-efficacy across 5 time points (baseline, quite date, 4, 12 and 24 weeks). At study baseline, mean depression scores fell above the clinical cut off of 16 (M = 16.31; SD = 13.53) on the Centre for Epidemiological Studies - Depression (CES-D) scale and fell below the clinical cut off at 24 weeks post quit date (M = 13.36; SD = 10.62). Results of multi-level modeling indicated a significant linear reduction in depressive symptoms and a significant linear improvement in self-efficacy to refrain from smoking across study visits. These results suggest that positive change in mood and self-efficacy may be helpful for PLH who remain smoke-free during a quit attempt.

Patient perspectives on the implementation of routinised syphilis screening with HIV viral load testing: Qualitative process evaluation of the Enhanced Syphilis Screening Among HIV-positive Men trial.

BACKGROUND: Syphilis infections have been on the rise, affecting men living with HIV in urban centres disproportionately. Since individuals in HIV care undergo routine blood testing, HIV clinics provide practical opportunities to conduct regular and frequent syphilis testing. Following the implementation of a routine syphilis testing intervention in HIV outpatient clinics, we conducted a qualitative process evaluation of patient experiences to measure patient acceptability, barriers to implementation, and facilitators of successful uptake. METHODS: Upon completion of the trial, which took place at four HIV outpatient clinics in Toronto and Ottawa, Canada, we recruited male patients attending these clinics from November 2017 to April 2018. Interviews were conducted on-site and were audio-recorded and transcribed verbatim. All participants provided written informed consent. Interview data were analyzed using grounded theory, assessing qualitative modulators of effective uptake of routinised syphilis testing. RESULTS: A total of 21 male patients were interviewed. Overall, interviewees found the clinical intervention acceptable, endorsing the practice of routinising syphilis testing alongside regular viral load bloodwork. Some men preferred, based on their self-assessment of syphilis risk, to opt out of testing; we considered this as a potential barrier to uptake of population-wide routinised syphilis testing. Interviewees also identified multiple facilitators of successful uptake, including the de-stigmatising of STI testing as a consequence of the universal nature of routinised testing. Participants recommended a routinised syphilis screening intervention to give patients peace of mind surrounding their sexual health. Participants identified HIV care clinics as comfortable and efficient locations to offer testing. CONCLUSIONS: Overall, most men were in support of implementing routinised syphilis testing as part of standard HIV care. From the patient perspective, HIV care clinics are convenient places to be tested for syphilis, and the routine approach was viewed to have a de-stigmatisng effect on syphilis testing. TRIAL REGISTRATION: ClinicalTrials.gov NCT02019043; registered December 23, 2013.

Infection with antiretroviral-susceptible HIV in an individual adherent to pre-exposure prophylaxis: strategies for treatment initiation.

HIV pre-exposure prophylaxis (PrEP) is effective at preventing sexual acquisition of HIV, and failures in clinical trials are largely attributable to medication nonadherence. We report here a case of infection with a fully susceptible strain of HIV in an individual adherent to PrEP as demonstrated by pharmacy records and intracellular tenofovir diphosphate levels. At diagnosis, the viral load was 90 copies/mL precluding initial genotype testing due to low copy number. While PrEP failure is rare, this case underscores the importance of regular HIV testing for patient on PrEP and prompts discussion regarding the approach to treatment following failure where an initial genotype is not yet available or not possible due to low viral load. Few other case reports of PrEP failure exist in the literature and approaches to treatment varied widely. We suggest the initial viral copy number may guide next steps and discuss the risks and benefits of stopping PrEP, escalating therapy with integrase inhibitors or boosted protease inhibitors, or switching to non-nucleoside antiretroviral treatment regimens.

Knowledge and Attitudes of Canadian Cardiac Surgeons Regarding Patients With Human Immunodeficiency Virus.

BACKGROUND: Current data on cardiac surgical practices for people living with human immunodeficiency virus (HIV) are lacking. We hypothesized that cardiac surgeons would consider people living with HIV as candidates for the full scope of cardiac surgery, including heart transplant for these patients. METHODS: We conducted a prospective survey of 155 cardiac surgeons across Canada to evaluate their current clinical perceptions regarding cardiac surgery in people living with HIV. Specifically, we evaluated their assessment of eligibility toward a wide scope of cardiac surgeries by using representative clinical scenarios. RESULTS: A total of 63 surgeon responses (40.6%) were completed. The majority of surgeons agreed that a 50-year-old man with HIV and no other comorbidities, who had been receiving combination antiretroviral therapy for 5 years with an undetectable viral load since starting therapy and a CD4 count greater than 350 cells/µL, would be a candidate for valve replacement (73%), valve repair surgery (74.6%), or coronary artery bypass graft surgery (79.4%). Few surgeons believed that this patient would be eligible for cardiac transplantation (7.9%) or could be a cardiac transplant donor (1.6%). There was clinical equipoise over the eligibility for ventricular assist device surgery. CONCLUSIONS: A majority of cardiac surgeons would perform coronary artery bypass graft surgery or valve surgery on patients with controlled HIV, but most consider HIV status as a prohibitive risk factor for cardiac transplantation. Although this may represent an opportunity for continuing medical education for cardiac surgeons, it also highlights the need for contemporary, high-quality evidence in this patient population.

Low human papillomavirus (HPV) vaccine uptake among men living with human immunodeficiency virus (HIV): Cross-sectional findings from a clinical cohort.

Men living with human immunodeficiency virus (HIV) are internationally recognized as a priority population for human papillomavirus (HPV) vaccination. Our objective was to explore HPV vaccine uptake among men living with HIV in Ontario, Canada, and investigate differences between vaccinated and unvaccinated men. We used data from a cross-sectional questionnaire administered between 2016 and 2017 among men living with HIV and participating in the Ontario HIV Treatment Network Cohort Study. We calculated the proportion vaccinated against HPV, described vaccination experiences, and HPV vaccine knowledge, and calculated differences in characteristics between vaccinated and unvaccinated men. Among 1651 men (mean age = 51 years, 72% identified as gay), 7% were vaccinated (95% confidence interval[CI] 5.5-7.9%); 85% received their first dose at a primary care or HIV clinic. Among unvaccinated men, 40% were unaware of the HPV vaccine, 65% reported low perceived risk for HPV, and 8% discussed HPV vaccination with a physician. Compared to unvaccinated men, vaccinated men were younger, most identified as gay, had a higher education/income, reported a higher number of recent sex partners, and had a history of bacterial sexually transmitted infections (STIs), HPV, anogenital warts, and/or anal cancer. Our findings reveal that few men living with HIV were vaccinated against HPV. This may be influenced by low HPV awareness, prohibitive cost, and lack of physician recommendation. Several men reporting lower socio-economic status, older men, and heterosexual, bisexual, and other men who have sex with men were missed for vaccination. Primary care and HIV clinics may be ideal locations to increase uptake.

Vedolizumab treatment across antiretroviral treatment interruption in chronic HIV infection: the HAVARTI protocol for a pilot dose-ranging clinical trial to assess safety, tolerance, immunological and virological activity.

INTRODUCTION: Continuous antiretroviral therapy (ART) suppresses HIV plasma viral load (pVL) to very low levels, which allows for some immune recovery. Discontinuation of ART leads to pVL rebound from reservoirs of persistence and latency, and progressive immunodeficiency. One promising but controversial strategy targeting CD4+ T lymphocytes with a monoclonal antibody (mAb) against α4ß7 integrin has shown promise through sustained virological remission of pVL (SVR) in SIV239-infected rhesus macaques. We propose to assess the safety and tolerability of vedolizumab, a licensed humanised mAb against human α4ß7 integrin, in healthy HIV-infected adults on ART. This study will also assess, by analytical treatment interruption (ATI), whether vedolizumab treatment can induce SVR beyond ART and vedolizumab treatment. METHODS AND ANALYSIS: The HIV-ART-vedolizumab-ATI (HAVARTI) trial is a single-arm, dose-ranging pilot trial in healthy HIV-positive adult volunteers receiving ART. Twelve consenting persons will be enrolled in sequential groups of 4 to each serial dosing vedolizumab regimen (300 mg, 150 mg, 75 mg). The primary outcomes are: (1) to assess the safety and tolerability of seven serial infusions of vedolizumab at each of three doses; (2) to identify the immunovirological measures, including pVL and T-cell kinetics, that characterise HIV/ART cases before, during, after vedolizumab treatment and ATI; and (3) to seek SVR of pVL after ATI. Secondary outcomes will include immune reconstitution and pVL suppression as well as immune reconstitution and long-term safety following re-initiation of ART in the absence of SVR. ETHICS AND DISSEMINATION: The study protocol was approved by the Ottawa Health Science Network-REB and by the Health Canada Therapeutic Products Directorate. A Data Safety Monitor will review safety information at regular intervals. The final manuscript will be submitted to an open access journal within a year of study completion. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT03147859; https://clinicaltrials.gov/ct2/show/NCT03147859.

The Canadian HIV and aging cohort study - determinants of increased risk of cardio-vascular diseases in HIV-infected individuals: rationale and study protocol.

BACKGROUND: With potent antiretroviral drugs, HIV infection is becoming a chronic disease. Emergence of comorbidities, particularly cardiovascular disease (CVD) has become a leading concern for patients living with the infection. We hypothesized that the chronic and persistent inflammation and immune activation associated with HIV disease leads to accelerated aging, characterized by CVD. This will translate into higher incidence rates of CVD in HIV infected participants, when compared to HIV negative participants, after adjustment for traditional CVD risk factors. When characterized further using cardiovascular imaging, biomarkers, immunological and genetic profiles, CVD associated with HIV will show different characteristics compared to CVD in HIV-negative individuals. METHODS/DESIGN: The Canadian HIV and Aging cohort is a prospective, controlled cohort study funded by the Canadian Institutes of Health Research. It will recruit patients living with HIV who are aged 40 years or older or have lived with HIV for 15 years or more. A control population, frequency matched for age, sex, and smoking status, will be recruited from the general population. Patients will attend study visits at baseline, year 1, 2, 5 and 8. At each study visit, data on complete medical and pharmaceutical history will be captured, along with anthropometric measures, a complete physical examination, routine blood tests and electrocardiogram. Consenting participants will also contribute blood samples to a research biobank. The primary outcome is incidence of a composite of: myocardial infarction, coronary revascularization, stroke, hospitalization for angina or congestive heart failure, revascularization or amputation for peripheral artery disease, or cardiovascular death. Preplanned secondary outcomes are all-cause mortality, incidence of the metabolic syndrome, incidence of type 2 diabetes, incidence of renal failure, incidence of abnormal bone mineral density and body fat distribution. Patients participating to the cohort will be eligible to be enrolled in four pre-planned sub-studies of cardiovascular imaging, glucose metabolism, immunological and genetic risk profile. DISCUSSION: The Canadian HIV and Aging Cohort will provide insights on pathophysiological pathways leading to premature CVD for patients living with HIV.

An HIV-tailored quit-smoking counselling pilot intervention targeting depressive symptoms plus Nicotine Replacement Therapy.

Cardiovascular disease (CVD) rates among people living with HIV/AIDS (PHAs) are high. Rates of cigarette smoking, a leading contributor to CVD among PHAs, are 40-70% (2-3 times higher than the general population). Furthermore, PHAs have high rates of depression (40-60%), a risk factor for smoking cessation relapse. The current pilot study examined the effectiveness of a specifically tailored 5-session smoking cessation counselling programme for PHAs, which addressed depression, in combination with Nicotine Replacement Therapy (NRT) in a cohort of PHA smokers (n = 50). At 6-month follow-up, 28% of participants demonstrated biochemically verified abstinence from smoking. This result compares favourably to other quit-smoking intervention studies, particularly given the high percentage of HIV+ smokers with depression. At study baseline, 52% of HIV+ smokers scored above the clinical cut-off for depression on the Centre for Epidemiological Studies - Depression (CES-D) scale. HIV+ smokers with depression at study baseline demonstrated quantitatively lower depression at 6-month follow-up with a large effect size (d = 1), though it did not reach statistical significance (p = .058). Furthermore, those with depression were no more likely to relapse than those without depression (p = .33), suggesting that our counselling programme adequately addressed this significant barrier to smoking cessation among PHAs. Our pilot study indicates the importance of tailored programmes to help PHAs quit smoking, the significance of addressing depressive symptoms, and the need for tailored counselling programmes to enhance quit rates among PHAs.

Transcriptional regulation of the IL-7Rα gene by dexamethasone and IL-7 in primary human CD8 T cells.

Interleukin-7 is essential for the development and maintenance of T cells, and the expression of the IL-7 receptor is tightly regulated at every stage of the T cell's lifespan. In mature CD8 T cells, IL-7 plays important roles in cell survival, peripheral homeostasis, and cytolytic function. The IL-7 receptor alpha-chain (CD127) is expressed at high levels on naïve and memory cells, but it is rapidly downregulated upon IL-7 stimulation. In this study, we illustrate the dynamicity of the CD127 promoter and show that it possesses positive as well as negative regulatory sites involved in upregulating and downregulating CD127 expression, respectively. We cloned the CD127 gene promoter and identified key cis-regulatory elements required for CD127 expression in mature resting primary CD8 T cells. The core promoter necessary for efficient basal transcription is contained within the first 262 bp upstream of the TATA box. Additional positive regulatory elements are located between -1200 and -2406 bp, conferring a further 2- to 4-fold enhancement in gene expression. While transcription of the CD127 gene is increased directly through a glucocorticoid response element located between -2255 and -2269 bp upstream of the TATA box, we identified a suppressive region that lies upstream of 1760 bp from the TATA box, which is likely involved in the IL-7-mediated suppression of CD127 transcription. Finally, we illustrated IL-7 does not bias alternative splicing of CD127 transcripts in primary human CD8 T cells.

Suppressor of cytokine signaling (SOCS) proteins are induced by IL-7 and target surface CD127 protein for degradation in human CD8 T cells.

Given the essential role interleukin (IL)-7 plays in T-cell survival, homeostasis and function, it is no surprise expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. We have previously shown IL-7 binding to its receptor on the surface of CD8 T cells leads to both suppression of CD127 gene transcription and loss of existing CD127 protein from the cell membrane. Indeed upon binding IL-7, CD127 is rapidly internalized into early endosomes where phosphorylation by JAK targets the receptor for degradation. We now show that IL-7 induces the expression of suppressor of cytokine signaling (SOCS) proteins CIS, SOCS1 and SOCS2 through the JAK/STAT-5 pathway and that CIS and SOCS2 specifically interact with CD127 in early endosomes and direct the receptor complex to the proteasome for degradation. These results illustrate how expression of the IL-7 receptor and thus IL-7 signaling is modulated in human CD8 T cells by a negative feedback mechanism dependent on members of the SOCS family of proteins.

Enhanced syphilis screening among HIV-positive men (ESSAHM): a study protocol for a clinic-randomized trial with stepped wedge design.

BACKGROUND: The current syphilis epidemic among urban men who have sex with men (MSM) has serious implications for those co-infected with human immunodeficiency virus (HIV). Routine and frequent syphilis screening has the potential to ensure early detection and treatment, minimize disease burden, and help control the ongoing spread of syphilis and HIV. We aim to enhance syphilis screening among HIV-positive men by conducting a clinic-based intervention that incorporates opt-out syphilis testing into routine HIV laboratory evaluation for this population. Trial objectives are to determine the degree to which the intervention (1) increases the detection rate of untreated syphilis, (2) increases screening coverage, (3) increases screening frequency, and (4) reaches men at highest risk according to sexual behaviors. METHODS/DESIGN: The trial is a pragmatic, stepped wedge cluster-randomized controlled trial that introduces the intervention stepwise across four urban HIV clinics in Ontario, Canada. The intervention includes standing orders for syphilis serological testing whenever a male in HIV care undergoes HIV viral load testing, which typically occurs every 3-6 months. The control condition is the maintenance of current, provider-initiated syphilis testing practice. Approximately 3100 HIV-positive men will be followed over 30 months. Test results will be obtained from the centralized provincial laboratory in Ontario and will be supplemented by a standardized clinical worksheet and medical chart review at the clinics. Detailed clinical, psychosocial, and behavioral data is available for a subset of men receiving HIV care who are also participants of the province-wide Ontario HIV Treatment Network Cohort Study. Process evaluation plans include audit and feedback of compliance of the participating centers to identify potential barriers to the introduction of this type of practice into routine care. Health economic components include evaluation of the impact and cost-effectiveness of the intervention. DISCUSSION: This trial will be the first of its kind in Canada and will provide evidence regarding the feasibility, clinical effectiveness, and cost-effectiveness of a clinic-based intervention to improve syphilis screening among HIV-positive men. Involvement of knowledge users in all stages of trial design, conduct, and analysis will facilitate scale-up should the intervention be effective. TRIAL REGISTRATION: ClinicalTrials.gov NCT02019043.

IL-7 induces clathrin-mediated endocytosis of CD127 and subsequent degradation by the proteasome in primary human CD8 T cells.

Interleukin-7 (IL-7), a key immunoregulatory cytokine, plays an essential role in peripheral T-cell homeostasis and function. Signaling via the IL-7 receptor is tightly regulated and we and others have shown IL-7 provides negative feedback on its own signaling by downregulating expression of the IL-7 receptor alpha-chain (CD127) through both suppression of CD127 gene transcription and by internalization of existing CD127 proteins from the cell membrane. We show here for the first time in primary human CD8 T cells that upon stimulation with IL-7, CD127 is internalized through clathrin-coated pits, a process dependent on both lipid-raft formation and the activity of dynamin. As visualized by confocal microscopy, CD127 shows increased co-localization with clathrin within 5 min of IL-7 stimulation and within 15-30 min is seen in multiple intracellular punctae co-localizing with the early endosomal marker EEA1. By 2 h after addition of IL-7, CD127 staining associates with the late endosomal marker RAB7 and with the proteasomal 20S subunit. By inducing receptor internalization and translocation from early endosomes to the proteasome, IL-7 directly influences its receptor density on the cell surface and thus regulates the intensity of its own signaling cascades. Given the important role IL-7 plays in T-cell development, homeostasis and function, deciphering how expression of its receptor is controlled on the cell surface is essential in understanding how T-cell activity can be regulated in different microenvironments and in response to different pathogens.

The N-Terminal Region of HIV-1 Tat Protein Binds CD127 in Human CD8 T Cells to Target the Receptor for Down Regulation Through Tat's Basic Region.

We have previously shown that soluble HIV-1 Tat protein down regulates surface expression of the interleukin (IL)-7 receptor alpha-chain (CD127) on human CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. Once taken up by CD8 T cells, Tat translocates to the inner leaflet of the plasma membrane where it interacts with the cytoplasmic tail of CD127 inducing receptor internalization and degradation by the proteasome. Here we characterized the regions of Tat required to interact with CD127 and induce receptor down regulation from the cell surface. To do this, a series of histidine-tagged Tat deletion mutants were generated and expressed as purified soluble protein, or cloned into a DNA expression vector and transfected into primary human CD8 T cells and a CD127 expressing Jurkat cell line. Protein-protein interactions were assessed by co-immunoprecipitation. Substitution of the first 10 Nterminal residues or deletion of residues 17-21 prevented Tat from interacting with and down regulating CD127 from the cell surface. Deletion of the basic region also prevented Tat from down regulating CD127 but did not prevent Tat from binding to the receptor. Notably, an endogenously expressed Tat variant lacking the basic region caused an accumulation of CD127 at the cell surface. We propose a model where Tat interacts with CD127 via its N-terminal region and recruits cellular factors via its basic region to down regulate CD127 from the cell surface.

IL-7 receptor recovery on CD8 T-cells isolated from HIV+ patients is inhibited by the HIV Tat protein.

Expression of the IL-7 receptor α-chain (CD127) is decreased on CD8 T-cells in HIV infected patients and partially recovers in those receiving antiretroviral therapy with sustained viral suppression. We have shown that soluble HIV Tat protein down regulates CD127 expression on CD8 T-cells isolated from healthy HIV-negative individuals. Tat is taken up by CD8 T-cells via endocytosis, exits the endosome and then translocates to the inner leaflet of the cell membrane where it binds to the cytoplasmic tail of CD127 inducing receptor internalization and degradation by the proteasome. This down regulation of CD127 by Tat results in impaired CD8 T-cell function. Interestingly, suppression of CD127 by Tat is reversible and requires the continual presence of Tat in the culture media. We thus questioned whether the low IL-7 receptor expression evident on CD8 T-cells in HIV+ patients was similarly reversible and if suppression of the receptor could be maintained ex vivo by Tat protein alone. We show here that when CD8 T-cells isolated from HIV+ patients are incubated alone in fresh medium, low CD127 expression on the cell surface recovers to normal levels. This recovery of CD127, however, is completely inhibited by the addition of HIV Tat protein to the culture media. This study then provides evidence that soluble factor(s) are responsible for low CD127 expression on circulating CD8 T-cells in HIV+ individuals and further implicates Tat in suppressing this receptor essential to CD8 T-cell proliferation and function.

Jak/STAT and PI3K signaling pathways have both common and distinct roles in IL-7-mediated activities in human CD8+ T cells.

IL-7 plays an important role in T cell survival, function, and memory cell development, yet the role of cytokine signaling pathways in these processes has not been fully elucidated. Moreover, the underlying mechanisms for the observed impairment of IL-7 activity in diseases, such as HIV infection, breast cancer, and autoimmunity, are not well understood. It was therefore hypothesized that IL-7-induced signaling molecules could be linked with distinct IL-7-associated activities. To address this, the activation and functional associations of IL-7-induced signaling pathways, specifically antigen-independent activities that are relevant to T cell homeostasis, were examined. Low concentrations of IL-7 (100 pg/ml) are capable of activating the Jak-STAT and PI3K signaling pathways, whereas higher concentrations (500-1000 pg/ml) were required to induce Bcl-2 production and glucose uptake. Even higher concentrations of IL-7 (10,000 pg/ml) were needed to induce cell proliferation and intracellular accumulation of perforin. Inhibition of Jak activation reduced IL-7-induced Bcl-2 and perforin production, whereas inhibition of Jak/STAT or PI3K pathways reduced glucose uptake and proliferation. This study suggests a complex control of IL-7-associated activities in the absence of antigen stimulation. These data may provide insights into mechanisms of impaired IL-7 signaling and function in disease and could be relevant for the study of IL-7-based immunotherapeutics. Specifically, this study has linked STAT5 and PI3K activation to shared and distinct IL-7-associated activities in human CD8+ T cells.