T-cell Senescence and T-cell Activation Predict Carotid Atherosclerosis in HIV-infected Women
*******NATAP CROI POSTER OF THE YEAR 2010******
Reported by Jules Levin
CROI 2010 Feb 16-19 SF
Robert C Kaplan1, PhD, Elizabeth Sinclair2, PhD, Alan L Landay3, PhD, Nell Lurain3, PhD, Stephen J Gange4, PhD, Richey Sharrett4, MD, DrPH, Xiaonan Xue1, PhD, Peter Hunt2, MD, Howard N Hodis5, MD, Steven g Deeks,2, MD
Affiliations: 1Albert Einstein College of medicine, 2Univ of California-SF, 3Rush University Medical Center, 4Johns Hopkins Bloomberg School og Public Health, 5Univ of Southern California
Collectively, these observations are consistent with a model in which untreated HIV infection results in immune activation, accelerated immunologic aging and the emergence of a population of potentially dysfunctional immunosenescent T cells. Antiretroviral therapy-mediated suppression of HIV replication may only partially reverse or prevent this process. The presence of a large population of activated and/or senescent T cells may be causally associated with the premature onset of CVD.
SUMMARY OF RESULTS/DISCUSSION
Consistent with prior data, HIV infection was associated with markedly elevated levels of activated (CD38+HLA-DR+) peripheral T-cells. Viremic suppression through effective antiretroviral therapy appeared to reduce but not completely reverse this process.
Among HIV-infected women, T-cell activation was associated with markers of subclinical carotid artery disease after adjustment for multiple confounders.
Among HIV-infected women, CD8+ T-cell senescence, and to a lesser extent CD4+ T-cell senescence (phenotypically defined by absence of CD28 and presence of CD57), were also associated with HIV disease and with the presence subclinical carotid artery disease.
These associations of T-cell activation and senescence with carotid artery parameters were not observed in a population of HIV-uninfected controls who were studied using identical methods and who had comparable cardiovascular risk factor profiles. Relatively small sample size of the HIV-uninfected group is a limitation.
HIV infection results in chronic elevated T cell activation and inflammation. This inflammatory state is reduced but not normalized by antiretrovirial therapy. Among treated patients, inflammatory markers are associated with risk of death and cardiovascular events. In HIV seronegative individuals, inflammation may be associated with accelerated immunologic aging (immunosenescence). The degree to which immunosenescence exists during treated HIV infection, and the impact of this process on cardiovascular disease has not been defined.
HIV infected (n=115) and matched HIV uninfected (n=43) participants in the Women’s Interagency HIV Study underwent B-mode carotid ultrasound. Measurements included carotid artery distensibility and carotid intima-media thickness (IMT), with carotid lesions defined as IMT >1,5mm. T-cell activation (CD38+HLA-DR+) and T-cell senescence (CD57+CD28-) were measured using flow cytometry.
(from Jules: Carotid distensibility (CD) is a measure of carotid artery elasticity that has been introduced as a risk factor for cardiovascular disease. Morphological studies have indicated that arterial distensibility depends on different factors such as blood pressure and age. Aging is the main variable responsible for functional changes in the arterial wall leading to an increase in arterial stiffness)
The mean age of the HIV-infected and unifected women were 46.6 and 47.2 years, respectively. Most subjects were on HAART (n=66) and many had undetectable viral loads (n=28).
As compared to the HIV uninfected subjects, the treated women with undetectable viral loads had higher levels of CD4 T-cell activation (p<.01) and CD8 T-cell activation (p<.02); CD8+ T cell senescence (p=0.07) but not CD4+ T cell senescence (p=0.53) also tended to be increased in the treated aviremic women.
Carotid artery distensibility was decreased among the HIV-infected group (p=0.01). Among HIV-infected women, higher CD4+ activation (p<0.01) and CD8+ activation (p=0.02) were significantly associated with reduced carotid artery distensibility, independent of age, CD4 count, and viral load.
CD4+ senescence (p=0.01) and CD8+ senescence (P=0.01) were also associated with reduced carotid artery distensibility among HIV-infected women.
Similarly, carotid lesions were significantly increased among HIV-infected women with CD4+ T-cell count<200 as compared with controls, and among the HIV-infected subjects there was an association of T-cell activation and senescence with the presence of carotid lesions.
R-cell CD38+ expression, a marker of immune activation, predicts early and late HIV disease course (Giorgi JID 1999).
Immune activation predicts HIV prognosis independent of viral load and may be one mechanism contributing to CD4+ T-cell loss during untreated HIV, or inability to reconstitute peripheral CD4+ T cells during treated HIV.
Activated T-cells are present in artherosclerotic plaque and may contribute to CVD (CD4+, possibly CD8+) (Hansson Am J Path 1989).
Aging is associated with emergence of senescent cells, typically defined as long-lived, apoptosis-resistant cells that have limited proliferative capacity and often have a secretory phenotype (Campisi Nature 2009). Senescence is stimulated by environmental stress, inflammation, and genetic instability (mutations, epigenetic changes).
Senescent R-cells (defined by the loss of CD28 and/or presence of CD57) may relate to HIV disease stage, inflammatory response, and CVD (Liuzzo JACC 2007).
Given the evidence that HIV infection may be associated with premature onset of age-related diseases, we investigated the effect of untreated and treated HIV on T-cell immunosenescence. We also investigated the impact of T-cell activation and senescence on subclinical carotid artery disease measured by B-mode ultrasound.
The study was conducted among participants in the Women’s Interagency HIV Study (WIHS), prospective multicenter study conducted at 6 US centers.
Characteristics of HIV-infected and HIV-uninfected women.
*over duration of study enrollment. For subjects who entered the study on HAART, peak plasma HIV RNA and nadir CD4 counts were also obtained by chart review.
3a. Results: T-cell activation and senescence in relation to HIV disease stage
3b. Results: CD4+ and CD8+ T-cell activation in relation to subclinical carotid artery disease
Among HIV+ women, higher expression of activation markers on CD4+ (left panel) and CD8+ (right panel) T-cells was associated with carotid lesions.
Interaction terms suggested that higher T-cell activation was associated with carotid lesions in HIV+, but not in HIV- (p = 0.061 for CD4+, p = 0.023 for CD8+).
Among HIV-infected women, higher CD4+ (left panel) and CD8+ (right panel) senescence were associated with reduced carotid artery distensibility.
3c. Results: Multivariate analyses of T-cell activation and senescence in relation to subclinical carotid artery disease
Among HIV-infected women, associations between T-cell activation and senescence and subclinical carotid artery disease persisted after adjustment for HIV-related and CVD-related covariates.
Shown at left are models adjusted for age and use of classes of antiretroviral medications. Results were unaffected by further adjustment for CVD risk factors, VL, and socioeconomics.
Associations are shown as PR or ß per SD change in activation/senescence.
cIMT, carotid intima-media thickness of the right common carotid artery
6. Detailed Methods
Carotid ultrasound: High resolution B-mode carotid artery ultrasound was used to image the far wall of the right common carotid artery (CCA), the internal carotid artery, and the carotid bifurcation according to the procedure of Hodis and colleagues. Right distal CCA intima-media thickness was measured (cIMT). The presence of carotid lesions was defined as a focal intima-media thickness >1.5 mm in any of the imaged segments. To estimate arterial distensibility we obtained right CCA diameters at systole (DS) and diastole (DD) and brachial artery pulse pressure (PP); lower distensibility values reflect a stiffer artery. Standardized carotid artery ultrasound images were centrally measured by automated computerized edge detection using an in-house developed software package (Prowin, Patent, 2005, 2006). Laboratory assays: HIV infection was determined via serologic testing using enzyme-linked immunosorbent assay (ELISA) and confirmed using Western blot assays. Plasma HIV viral load was quantified using nucleic acid sequence based amplification commercial assays with a lower limit of quantification of 80 copies/mL (bioMérieux, Boxtel, NC), and total peripheral CD4+ T-cell counts were measured with standard flow cytometric methods, at laboratories participating in the National Institutes of Health/National Institute of Allergy and Infectious Disease Flow Cytometry Quality Assessment Program. T-cell activation and senescence were measured by immunophenotyping performed at the UCSF Core Immunology Laboratory, using methods that have been optimized and validated for frozen peripheral blood mononuclear cells (PBMCs). Cryopreserved PBMCs were rapidly thawed in warm media, washed, stained with Viacount (Millipore, Billerica, MA) and run on a Guava PCA (Millipore) to determine cell number and viability. Samples with viability of less than 40% were not analyzed. The mean PBMC viability was 75.0% in HIV-infected and 77.5% in HIV-uninfected women. PBMCs were stained for Aqua Amine Reactive Dye (Invitrogen, Carlsbad, CA) to exclude non-viable cells, and for surface expression of CD3, CD28 (BD Pharmingen, San Diego CA), CD4, CD38, HLA-DR (BD Biosciences, San Jose, CA), CD8 (Invitrogen) and CD57 (Biolegend, San Diego, CA). Stained cells were run on a customized BD LSR II and data analyzed using Flow Jo (Tree Star, Ashland, OR) to quantitate CD4+ and CD8+ T-cells expressing activation (CD38, HLA-DR) and senescence (CD28-, CD57+) markers.
Funding: Funded by the National Institute of Allergy and Infectious Diseases (UO1-AI-35004, UO1-AI-31834, UO1-AI-34994, UO1-AI-34989, UO1-AI-34993, and UO1-AI-42590) and by the National Institute of Child Health and Human Development (UO1-HD-32632), and co-funded by the National Cancer Institute, the National Institute on Drug Abuse, the National Institute on Deafness and Other Communication Disorders, the National Center for Research Resources (UCSF-CTSI Grant Number UL1 RR024131), the National Heart, Lung and Blood Institute (1R01HL095140, 1R01HL083760 to Albert Einstein), NIH/NIAID funding to the UCSF-GIVI Center for AIDS Research (P30AI027763) and NCR funding to the UCSF Clinical and Translational Science Institute (UL1RR024131).