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1.  Early HLA-B*57-Restricted CD8+ T Lymphocyte Responses Predict HIV-1 Disease Progression 
Journal of Virology  2012;86(19):10505-10516.
Although HLA-B*57 (B57) is associated with slow progression to disease following HIV-1 infection, B57 heterozygotes display a wide spectrum of outcomes, including rapid progression, viremic slow progression, and elite control. Efforts to identify differences between B57-positive (B57+) slow progressors and B57+ rapid progressors have largely focused on cytotoxic T lymphocyte (CTL) phenotypes and specificities during chronic stages of infection. Although CTL responses in the early months of infection are likely to be the most important for the long-term rate of HIV-1 disease progression, few data on the early CTL responses of eventual slow progressors have been available. Utilizing the Multicenter AIDS Cohort Study (MACS), we retrospectively examined the early HIV-1-specific CTL responses of 14 B57+ individuals whose time to development of disease ranged from 3.5 years to longer than 25 years after infection. In general, a greater breadth of targeting of epitopes from structural proteins, especially Gag, as well as of highly conserved epitopes from any HIV-1 protein, correlated with longer times until disease. The single elite controller in the cohort was an outlier on several correlations of CTL targeting and time until disease, consistent with reports that elite control is typically not achieved solely by protective HLA-mediated CTLs. When targeting of individual epitopes was analyzed, we found that early CTL responses to the IW9 (ISPRTLNAW) epitope of Gag, while generally subdominant, correlated with delayed progression to disease. This is the first study to identify early CTL responses to IW9 as a correlate of protection in persons with HLA-B*57.
PMCID: PMC3457253  PMID: 22811521
2.  Acute HIV-1 Seroconversion with an Unusual Plasma Biomarker Profile 
Clinical and Vaccine Immunology : CVI  2013;20(11):1774-1777.
An unusual case of acute primary HIV-1 infection in a man with a high plasma viral load, a 51-fold increase in C-reactive protein, and antibodies against only gp160 is described. Numerous serum cytokine concentrations were elevated during HIV-1 seroconversion.
PMCID: PMC3837784  PMID: 24006141
3.  A Challenge for the Future: Aging and HIV Infection 
Immunologic research  2010;48(1-3):59-71.
Older individuals (≥ 50 years of age) are increasingly becoming a new at-risk group for HIV-1 infection and, together with those surviving longer due to the introduction of anti-retroviral therapy (ART), it is predicted that more than half of all HIV-1-infected individuals in the U.S. will be greater than 50 years of age in the year 2015. Older individuals diagnosed with HIV-1 are prone to faster disease progression and reduced T-cell reconstitution despite successful virologic control with anti-retroviral therapy (ART). There is also growing evidence that the T-cell compartment in HIV-1+ adults displays an aged phenotype and HIV-1-infected individuals are increasingly diagnosed with clinical conditions more commonly seen in older uninfected persons. As aging in the absence of HIV infection is associated with alterations in T-cell function and immunosenescence, the combined impact of both HIV-1 infection and aging may provide an explanation for poorer clinical outcomes observed in older HIV-1-infected individuals. Thus, the development of novel therapeutics to stimulate immune function and delay immunosenescence is critical and would be beneficial to both the elderly and HIV-1 infected individuals.
PMCID: PMC3077114  PMID: 20734158
HIV; ART; CD4+ T-cells; CD8+ T-cells; IL-7; TAT2; aging
4.  Homeostasis of the Naive CD4+ T Cell Compartment during Aging1 
Despite thymic involution, the number of naive CD4+ T cells diminishes slowly during aging, suggesting considerable peripheral homeostatic expansion of these cells. To investigate the mechanisms behind, and consequences of, naive CD4+ T cell homeostasis, we evaluated the age-dependent dynamics of the naive CD4+ T cell subsets CD45RA+CD31+ and CD45RA+CD31−. Using both a cross-sectional and longitudinal study design, we measured the relative proportion of both subsets in individuals ranging from 22 to 73 years of age and quantified TCR excision circle content within those subsets as an indicator of proliferative history. Our findings demonstrate that waning thymic output results in a decrease in CD45RA+CD31+ naive CD4+ T cells over time, although we noted considerable individual variability in the kinetics of this change. In contrast, there was no significant decline in the CD45RA+CD31− naive CD4+ T cell subset due to extensive peripheral proliferation. Our longitudinal data are the first to demonstrate that the CD45RA+CD31+CD4+ subset also undergoes some in vivo proliferation without immediate loss of CD31, resulting in an accumulation of CD45RA+CD31+ proliferative offspring. Aging was associated with telomere shortening within both subsets, raising the possibility that accumulation of proliferative offspring contributes to senescence of the naive CD4+ T cell compartment in the elderly. In contrast, we observed retention of clonal TCR diversity despite peripheral expansion, although this analysis did not include individuals over 65 years of age. Our results provide insight into naive CD4+ T cell homeostasis during aging that can be used to better understand the mechanisms that may contribute to immunosenescence within this compartment.
PMCID: PMC2940825  PMID: 18209045
5.  Acceleration of Age-Associated Methylation Patterns in HIV-1-Infected Adults 
PLoS ONE  2015;10(3):e0119201.
Patients with treated HIV-1-infection experience earlier occurrence of aging-associated diseases, raising speculation that HIV-1-infection, or antiretroviral treatment, may accelerate aging. We recently described an age-related co-methylation module comprised of hundreds of CpGs; however, it is unknown whether aging and HIV-1-infection exert negative health effects through similar, or disparate, mechanisms. We investigated whether HIV-1-infection would induce age-associated methylation changes. We evaluated DNA methylation levels at >450,000 CpG sites in peripheral blood mononuclear cells (PBMC) of young (20-35) and older (36-56) adults in two separate groups of participants. Each age group for each data set consisted of 12 HIV-1-infected and 12 age-matched HIV-1-uninfected samples for a total of 96 samples. The effects of age and HIV-1 infection on methylation at each CpG revealed a strong correlation of 0.49, p<1 x10-200 and 0.47, p<1x10-200. Weighted gene correlation network analysis (WGCNA) identified 17 co-methylation modules; module 3 (ME3) was significantly correlated with age (cor=0.70) and HIV-1 status (cor=0.31). Older HIV-1+ individuals had a greater number of hypermethylated CpGs across ME3 (p=0.015). In a multivariate model, ME3 was significantly associated with age and HIV status (Data set 1: βage= 0.007088, p=2.08 x 10-9; βHIV= 0.099574, p=0.0011; Data set 2: βage= 0.008762, p=1.27x 10-5; βHIV= 0.128649, p= 0.0001). Using this model, we estimate that HIV-1 infection accelerates age-related methylation by approximately 13.7 years in data set 1 and 14.7 years in data set 2. The genes related to CpGs in ME3 are enriched for polycomb group target genes known to be involved in cell renewal and aging. The overlap between ME3 and an aging methylation module found in solid tissues is also highly significant (Fisher-exact p=5.6 x 10-6, odds ratio=1.91). These data demonstrate that HIV-1 infection is associated with methylation patterns that are similar to age-associated patterns and suggest that general aging and HIV-1 related aging work through some common cellular and molecular mechanisms. These results are an important first step for finding potential therapeutic targets and novel clinical approaches to mitigate the detrimental effects of both HIV-1-infection and aging.
PMCID: PMC4373843  PMID: 25807146
6.  Comparison of Antibodies That Mediate HIV Type 1 gp120 Antibody-Dependent Cell-Mediated Cytotoxicity in Asymptomatic HIV Type 1-Positive Men and Women 
Recent studies suggest that HIV-specific antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies contribute to protective immunity against HIV. An important characteristic of future HIV vaccines will, therefore, be the ability to stimulate production of these antibodies in both men and women. Early studies suggest that men may have a better ADCC antibody response against HIV than women. Our objective was to determine whether men and women differ with respect to their ADCC response to HIV-1 gp120. HIV-positive, asymptomatic untreated men and women were matched for race, age, CD4+ T cell number, HIV-1 viral load, and treatment and HIV-1 gp120 ADCC antibody titers were compared. A standard 51Cr-release assay was used to determine HIV-1 gp120 ADCC antibody titers in HIV-1-seropositive individuals from the Multicenter AIDS Cohort Study (MACS; n=32) and the Women's Interagency HIV Study (WIHS; n=32). Both sexes had high ADCC titers against HIV-1 gp120: 34.4% (n=11) and 40.6% (n=13) of men and women, respectively, had titers of 10,000; 62.5% (n=20) and 56.3% (n=18) had titers of 100,000. Groups did not differ in percent specific release (% SR), lytic units (LU), correlations of titer to viral load, or titer to CD4+ T cells in men or women. Both groups also had similar cross-clade ADCC antibody responses (p>0.5 for % SR and LU). Comparable groups of asymptomatic HIV-1-infected men and women had comparable HIV-1 gp120 ADCC antibodies. Both sexes had significant cross-clade reactivity. Differences between men and women may become evident as disease progresses; this should be evaluated at later stages of HIV-1 infection.
PMCID: PMC3887406  PMID: 23972002
7.  Assessing Immunophenotyping Performance: Proficiency-Validation for Adopting Improved Flow Cytometry Methods 
The continuous improvement and evolution of immune cell phenotyping requires periodic upgrading of laboratory methods and technology. Flow cytometry laboratories that are participating in research protocols sponsored by the NIAID are required to perform “switch” studies to validate performance before methods for T-cell subset analysis can be changed.
Switch studies were conducted among the four flow cytometry laboratories of the Multicenter AIDS Cohort Study (MACS), comparing a 2-color, lyse-wash method and a newer, 3-color, lyse no-wash method. Two of the laboratories twice failed to satisfy the criteria for acceptable differences from the previous method. Rather than repeating more switch studies, these laboratories were allowed to adopt the 3-color, lyse no-wash method. To evaluate the impact of the switch to the new method at these two sites, their results with the new method were evaluated within the context of all laboratories participating in the NIH-NIAID-Division of AIDS Immunology Quality Assurance (IQA) proficiency-testing program.
Laboratory performance at these two sites substantially improved relative to the IQA standard test results. Variation across the four MACS sites and across replicate samples was also reduced.
Although switch studies are the conventional method for assessing comparability of laboratory methods, two alternatives to the requirement of repeating failed switch studies should be considered: (1) test the new method and assess performance on the proficiency testing reference panel, and (2) prior to adoption of the new methods, use both the old and the new method on the reference panel samples and demonstrate that performance with the new method is better according to standard statistical procedures. These alternatives may help some laboratories’ transition to a new and superior methodology more quickly than if they are required to attempt multiple, serial switch studies.
PMCID: PMC4100219  PMID: 17205569
HIV; immunophenotyping; flow cytometry; quality assessment; multicenter studies
8.  Differential Blood and Mucosal Immune Responses against an HIV-1 Vaccine Administered via Inguinal or Deltoid Injection 
PLoS ONE  2014;9(2):e88621.
Mucosal immunity is central to sexual transmission and overall pathogenesis of HIV-1 infection, but the ability of vaccines to induce immune responses in mucosal tissue compartments is poorly defined. Because macaque vaccine studies suggest that inguinal (versus limb) vaccination may better target sexually-exposed mucosa, we performed a randomized, double-blinded, placebo-controlled Phase I trial in HIV-1-uninfected volunteers, using the recombinant Canarypox (CP) vaccine vCP205 delivered by different routes. 12 persons received vaccine and 6 received placebo, divided evenly between deltoid-intramuscular (deltoid-IM) or inguinal-subcutaneous (inguinal-SC) injection routes. The most significant safety events were injection site reactions (Grade 3) in one inguinal vaccinee. CP-specific antibodies were detected in the blood of all 12 vaccinees by Day 24, while HIV-1-specific antibodies were observed in the blood and gut mucosa of 1/9 and 4/9 evaluated vaccinees respectively, with gut antibodies appearing earlier in inguinal vaccinees (24–180 versus 180–365 days). HIV-1-specific CD8+ T lymphocytes (CTLs) were observed in 7/12 vaccinees, and blood and gut targeting were distinct. Within blood, both deltoid and inguinal responders had detectable CTL responses by 17–24 days; inguinal responders had early responses (within 10 days) while deltoid responders had later responses (24–180 days) in gut mucosa. Our results demonstrate relative safety of inguinal vaccination and qualitative or quantitative compartmentalization of immune responses between blood and gut mucosa, and highlight the importance of not only evaluating early blood responses to HIV-1 vaccines but also mucosal responses over time.
Trial Registration NCT00076817
PMCID: PMC3928250  PMID: 24558403
9.  Natural Killer T Cells in Advanced Melanoma Patients Treated with Tremelimumab 
PLoS ONE  2013;8(10):e76829.
A significant barrier to effective immune clearance of cancer is loss of antitumor cytotoxic T cell activity. Antibodies to block pro-apoptotic/downmodulatory signals to T cells are currently being tested. Because invariant natural killer T cells (iNKT) can regulate the balance of Th1/Th2 cellular immune responses, we characterized the frequencies of circulating iNKT cell subsets in 21 patients with melanoma who received the anti-CTLA4 monoclonal antibody tremelimumab alone and 8 patients who received the antibody in combination with MART-126–35 peptide-pulsed dendritic cells (MART-1/DC). Blood T cell phenotypes and functionality were characterized by flow cytometry before and after treatment. iNKT cells exhibited the central memory phenotype and showed polyfunctional cytokine production. In the combination treatment group, high frequencies of pro-inflammatory Th1 iNKT CD8+ cells correlated with positive clinical responses. These results indicate that iNKT cells play a critical role in regulating effective antitumor T cell activity.
PMCID: PMC3805549  PMID: 24167550
10.  Value of a Quality Assessment Program in Optimizing Cryopreservation of Peripheral Blood Mononuclear Cells in a Multicenter Study 
Cryopreservation of peripheral blood mononuclear cells (PBMC) allows assays of cellular function and phenotype to be performed in batches at a later time on PBMC at a central laboratory to minimize assay variability. The Multicenter AIDS Cohort Study (MACS) is an ongoing prospective study of the natural and treated history of human immunodeficiency virus (HIV) infection that stores cryopreserved PBMC from participants two times a year at four study sites. In order to ensure consistent recovery of viable PBMC after cryopreservation, a quality assessment program was implemented and conducted in the MACS over a 6-year period. Every 4 months, recently cryopreserved PBMC from HIV-1-infected and HIV-1-uninfected participants at each MACS site were thawed and evaluated. The median recoveries of viable PBMC for HIV-1-infected and -uninfected participants were 80% and 83%, respectively. Thawed PBMC from both HIV-1-infected and -uninfected participants mounted a strong proliferative response to phytohemagglutinin, with median stimulation indices of 84 and 120, respectively. Expression of the lymphocyte surface markers CD3, CD4, and CD8 by thawed PBMC was virtually identical to what was observed on cells measured in real time using whole blood from the same participants. Furthermore, despite overall excellent performance of the four participating laboratories, problems were identified that intermittently compromised the quality of cryopreserved PBMC, which could be corrected and monitored for improvement over time. Ongoing quality assessment helps laboratories improve protocols and performance on a real-time basis to ensure optimal cryopreservation of PBMC for future studies.
PMCID: PMC3623420  PMID: 23408528
11.  A Frailty-Related Phenotype Before HAART Initiation as an Independent Risk Factor for AIDS or Death After HAART Among HIV-Infected Men 
In the general population, frailty, a late stage of the aging process, predicts mortality. We investigated whether manifesting a previously defined frailty-related phenotype (FRP) before initiating highly active antiretroviral therapy (HAART) affects the likelihood of developing clinical AIDS or mortality after HAART initiation.
Among 596 HIV-infected men in the Multicenter AIDS Cohort Study whose date of HAART initiation was known within ±6 months and who had an assessable FRP status within 3 years before HAART, survival analyses were performed to assess the effect of FRP manifestation on clinical AIDS or death after HAART.
In men free of AIDS before HAART, AIDS or death after HAART occurred in 13/36 (36%) men who exhibited the FRP before HAART but only in 69/436 (16%) men who did not (hazard ratio = 2.6; 95% confidence interval = 1.4–4.6; p < .01). After adjusting for age, ethnicity, education, nadir CD4+ T-cell count, peak HIV viral load, and hemoglobin in the 3 years before HAART, having the FRP at >25% of visits in the 3 years before HAART significantly predicted AIDS or death (adjusted hazard ratio = 3.8; 95% confidence interval = 1.9–7.9; p < .01). Results were unchanged when the analysis was restricted to the 335 AIDS-free men who were HAART responders, to the 124 men who had AIDS at HAART initiation, or to the subsets of men for whom indices of liver and kidney function could be taken into account.
Having a persistent frailty-like phenotype before HAART initiation predicted a worse prognosis after HAART, independent of known risk factors.
PMCID: PMC3156632  PMID: 21719610
HIV; Aging; Frailty; HAART response; Survival analysis
12.  Increasing CTL Targeting of Conserved Sequences During Early HIV-1 Infection Is Correlated to Decreasing Viremia 
Early HIV-1 infection is marked by rapid evolution of both CD8+ T lymphocyte (CTL) epitope targeting and viral sequences, while chronic infection demonstrates relative stability of these parameters. To examine the interactions of changing CTL targeting and viremia in early infection, we assessed CTL targeting and viremia levels in persons during early HIV-1 infection (estimated 15–271 days post-infection) who were placed on effective antiretroviral therapy. Pre-therapy, CTL targeting of viral proteins varied between persons depending on time after infection. Across individuals, increasing time after infection was associated with increasing Gag and Pol targeting, suggesting increasing targeting of conserved sequences. The intensity of Gag targeting correlated to lower viremia levels, while Env targeting correlated to higher viremia levels during early infection. This suggested that shifted targeting towards more conserved sequences is involved with the drop of viremia during early infection, consistent with prior observations of correlation between Gag targeting and lower viremia during chronic infection. After suppressive antiretroviral therapy, CTL targeting was generally static, indicating that HIV-1 replication and evolution drives the evolution of CTL targeting in early infection. Overall, these data suggest that early CTL targeting is directed towards more variable epitopes, causing escape and re-targeting until more conserved epitopes are recognized stably in chronic infection. Circumventing this natural history by pre-targeting CTL against more conserved epitopes with a vaccine could minimize the initial period of viral escape and immune damage during acute infection, improving long-term containment of HIV-1.
PMCID: PMC3101083  PMID: 21087140
14.  Copy Number Variation of KIR Genes Influences HIV-1 Control 
PLoS Biology  2011;9(11):e1001208.
The authors that the number of activating and inhibitory KIR genes varies between individuals and plays a role in the regulation of immune mechanisms that determine HIV-1 control.
A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection.
Author Summary
There is marked intrinsic variation in the extent to which individuals are able to control HIV-1. We have identified a genetic copy number variable region (CNV) in humans that plays a significant role in the control of HIV-1. This CNV is located in the genomic region that encodes the killer cell immunoglobulin-like receptors (KIRs) and specifically affects the KIR3DS1 and KIR3DL1 genes, encoding two KIRs that interact with human leukocyte antigen B (HLA-B) ligands. KIRs are expressed on the surface of natural killer (NK) cells, which serve as important players in the innate immune response, and are involved in the recognition of infected and malignant cells through a loss or alteration in “self” ligands. We use both genetic association and functional evidence to show a strong interaction between KIR3DL1 and KIR3DS1, indicating that increasing gene counts for KIR3DL1 confer increasing levels of protection against HIV-1, but only in the presence of at least one copy of KIR3DS1. This effect was associated with a dramatic increase in the abundance of KIR3DS1+ NK cells in the peripheral blood, and strongly associated with a more robust capacity of peripheral NK cells to suppress HIV-1 replication in vitro. This work provides one of the few examples of an association between a relatively common CNV and a human complex trait.
PMCID: PMC3226550  PMID: 22140359
15.  Baseline Immune Phenotypes and CD4+ T Lymphocyte Responses to Antiretroviral Therapy in Younger versus Older HIV-infected Individuals 
Journal of clinical immunology  2011;31(5):873-881.
The purpose of the study was to determine associations between pre-antiretroviral therapy (ART) senescent CD8+ T lymphocytes and naïve versus non-naive CD8+ and CD4+ T lymphocyte subpopulations and CD4+ responses after initiation of ART in younger versus older individuals.
Retrospective analysis of 100 subjects with pre-ART cryopreserved peripheral blood mononuclear cells samples was performed with flow cytometry. Subjects were divided into four groups by age (30–50 years or >50 years) and 96-week CD4+ response (<100 or >200 cells/mm3). All subjects had 96-week viral suppression to <50 copies/ml. Regression was utilized to investigate associations between pre-ART CD8+ and CD4+ T cell phenotypes with age and CD4+ response categories.
Individuals <50 years had a lower frequency of senescent CD8+ T lymphocytes of the CD56+57+, CD56+, and CD28− phenotypes (95%CI −3.6 to −0.02; 95%CI −4.2 to −0.03; 95%CI −12.5 to −1.4, respectively) and a higher frequency of naïve (CD45RA+CD28+) CD8+ T lymphocytes (95%CI 2.6 to 10.9). Younger age and good CD4+ response were associated with a higher frequency of pre-ART naïve CD4+ T cells (95%CI 2.0 to 16.4 and 95% CI 1.5 to 15.6, respectively).
Prior to ART, younger HIV-infected individuals have a higher frequency of naïve CD4+ and CD8+ T cells and lower frequency of senescent CD8+ T cell phenotypes.
PMCID: PMC3194061  PMID: 21643890
HIV/AIDS; aging; immune risk phenotype; antiretroviral therapy; immune senescence
16.  Overexpression of MicroRNAs from the miR-17-92 Paralog Clusters in AIDS-Related Non-Hodgkin's Lymphomas 
PLoS ONE  2011;6(6):e20781.
Individuals infected by HIV are at an increased risk for developing non-Hodgkin's lymphomas (AIDS-NHL). In the highly active antiretroviral therapy (HAART) era, there has been a significant decline in the incidence of AIDS-associated primary central nervous system lymphoma (PCNSL). However, only a modest decrease in incidence has been reported for other AIDS-NHL subtypes. Thus, AIDS-NHLs remain a significant cause of morbidity and mortality in HIV infected individuals. Recently, much attention has been directed toward the role of miRNAs in cancer, including NHL. Several miRNAs, including those encoded by the miR-17-92 polycistron, have been shown to play significant roles in B cell tumorigenesis. However, the role of miRNAs in NHL in the setting of HIV infection has not been defined.
Methodology/Principal Findings
We used quantitative realtime PCR to assess the expression of miRNAs from three different paralog clusters, miR-17-92, miR-106a-363, and miR-106b-25 in 24 cases of AIDS-NHLs representing four tumor types, Burkitt's lymphoma (BL, n = 6), diffuse large B-cell lymphoma (DLBCL, n = 8), primary central nervous system lymphoma (PCNSL, n = 5), and primary effusion lymphoma (PEL, n = 5). We also used microarray analysis to identify a differentiation specific miRNA signature of naïve, germinal center, and memory B cell subsets from tonsils (n = 4). miRNAs from the miR-17-92 paralog clusters were upregulated by B cells, specifically during the GC differentiation stage. We also found overexpression of these miRNA clusters in all four AIDS-NHL subtypes. Finally, we also show that select miRNAs from these clusters (miR-17, miR-106a, and miR-106b) inhibited p21 in AIDS-BL and DLBCL cases, thus providing a mechanistic role for these miRNAs in AIDS-NHL pathogenesis.
Dysregulation of miR-17-92 paralog clusters is a common feature of AIDS-associated NHLs.
PMCID: PMC3116840  PMID: 21698185
17.  Comparison of Interlaboratory Variation in Absolute T-Cell Counts by Single-Platform and Optimized Dual-Platform Methods 
Previous studies have reported that the adoption of a single-platform flow cytometry cell counting method resulted in lower interlaboratory variation in absolute T cell counts as compared to predicate dual-platform flow cytometry methods which incorporate independent automated lymphocyte counts (Schnizlein-Bick et al., Clin Diagn Lab Immunol 2000;7:336–343; Reimann et al., Clin Diagn Lab Immunol 2000;7:344–351). In the present study, we asked whether use of a single-platform method could reduce variation in absolute cell counts across the laboratories in the Multicenter AIDS Cohort Study (MACS) (n = 4), as suggested by the studies cited.
Identical study samples were shipped overnight to the MACS laboratories either by the National Institute of Allergy and Infectious Diseases, Division of AIDS Immunology Quality Assessment (NIAID-IQA) proficiency-testing program (n = 14), or by the Los Angeles site of the MACS (n = 10). For each sample, two tubes of blood were received; one was used for an automated complete blood count and differential, and the other for flow cytometry. The latter was performed using both our current dual-platform method (three-color CD45 gating and automated hematology) and the single-platform method (with TruCOUNT® beads to generate the absolute counts).
The median percent coefficients of variation (%CVs) for the dual-platform and single-platform methods were 6.6 and 9.9, respectively, for CD4 T cell counts, and 5.9 and 8.5, respectively, for CD8 T cell counts (n = 24). These differences were not statistically significant. The differences in absolute T-cell counts between the MACS sites and the median of all laboratories participating in the NIAID-IQA were smaller for the dual-platform than for single-platform absolute count method.
In contrast to previous reports, we did not observe lower interlaboratory variation across the MACS sites for single-platform absolute lymphocyte subset counting relative to dual-platform methods. This result may be at least partly explained by the lower interlaboratory variation with the optimized dual-platform method in this study relative to the previous reports.
PMCID: PMC3086643  PMID: 19813263
HIV; absolute CD4 counts; flow cytometry; single platform; dual platform; interlaboratory variation
18.  The Relationship Between Antibody to R7V and Progression of HIV Type 1 Infection 
The presence of antibody to R7V (anti-R7VAb), a seven-amino acid sequence derived from β2-microglobulin incorporated into HIV-1 virions from the surface of infected cells, has been proposed as an early marker of nonprogressive HIV-1 infection. The present study was undertaken because no prospective studies have tested this hypothesis. Stored samples collected prospectively from 361 HIV-1 seroconverting men in the Multicenter AIDS Cohort Study (0.44–1.53 years after seroconversion) were assayed for the presence or absence of anti-R7VAb, using a standardized ELISA. Using Cox proportional hazards models, crude and adjusted relative hazards (RH) were determined for the following outcomes: (a) clinically defined AIDS, (b) clinically defined AIDS or CD4 T cell count of <200 cells/μl, and (c) death. A total of 143 (39.6%) men had early anti-R7VAb and 218 (60.4%) did not; 192 (53.2%) developed AIDS. At the visit tested, men with anti-R7VAb had significantly lower CD4 T cell counts and higher plasma HIV-1 viral loads than those without antibody. After adjustment for CD4 T cell count, HIV-1 viral load, CCR5 polymorphism, and use of combined antiretroviral therapy, the presence of anti-R7VAb was associated with a higher risk of progression for all outcomes, but not significantly so. Absence of anti-R7VAb was significantly associated with expression of HLA-B*5701 and -B*2705, two alleles associated with slower progression of HIV-1 disease. The early presence of anti-R7VAb in HIV-1 seroconverters was not associated with slower progression of HIV-1 disease.
PMCID: PMC2933163  PMID: 20415637
19.  The Dual Impact of HIV-1 Infection and Aging on Naïve CD4+ T-Cells: Additive and Distinct Patterns of Impairment 
PLoS ONE  2011;6(1):e16459.
HIV-1-infected adults over the age of 50 years progress to AIDS more rapidly than adults in their twenties or thirties. In addition, HIV-1-infected individuals receiving antiretroviral therapy (ART) present with clinical diseases, such as various cancers and liver disease, more commonly seen in older uninfected adults. These observations suggest that HIV-1 infection in older persons can have detrimental immunological effects that are not completely reversed by ART. As naïve T-cells are critically important in responses to neoantigens, we first analyzed two subsets (CD45RA+CD31+ and CD45RA+CD31-) within the naïve CD4+ T-cell compartment in young (20–32 years old) and older (39–58 years old), ART-naïve, HIV-1 seropositive individuals within 1–3 years of infection and in age-matched seronegative controls. HIV-1 infection in the young cohort was associated with lower absolute numbers of, and shorter telomere lengths within, both CD45RA+CD31+CD4+ and CD45RA+CD31-CD4+ T-cell subsets in comparison to age-matched seronegative controls, changes that resembled seronegative individuals who were decades older. Longitudinal analysis provided evidence of thymic emigration and reconstitution of CD45RA+CD31+CD4+ T-cells two years post-ART, but minimal reconstitution of the CD45RA+CD31-CD4+ subset, which could impair de novo immune responses. For both ART-naïve and ART-treated HIV-1-infected adults, a renewable pool of thymic emigrants is necessary to maintain CD4+ T-cell homeostasis. Overall, these results offer a partial explanation both for the faster disease progression of older adults and the observation that viral responders to ART present with clinical diseases associated with older adults.
PMCID: PMC3027697  PMID: 21298072
20.  Relationship between a frailty-related phenotype and progressive deterioration of the immune system in HIV-infected men 
Immunological similarities have been noted between HIV-infected individuals and older HIV-negative adults. Immunologic alterations with aging have been noted in frailty in older adults, a clinical syndrome of high risk for mortality and other adverse outcomes. Using a frailty-related phenotype (FRP), we investigated in the Multicenter AIDS Cohort Study (MACS) whether progressive deterioration of the immune system among HIV positive individuals independently predicts onset of FRP.
FRP was evaluated semiannually in 1,046 HIV-infected men from 1994–2005. CD4 T-cell count and plasma viral load were evaluated as predictors of FRP by logistic regression (GEE), adjusting for age, ethnicity, educational level, AIDS status, and treatment era (pre-HAART (1994–1995) and HAART (1996–1999 and 2000–2005)).
Adjusted prevalences of FRP remained low for CD4 T-cell counts >400 cells/mm3 and increased exponentially and significantly for lower counts. Results were unaffected by treatment era. After 1996, CD4 cell T-count, but not plasma viral load, was independently associated with FRP.
CD4 T-cell count predicted the development of a frailty-related phenotype among HIV infected men, independent of HAART use. This suggests that compromise of the immune system in HIV-infected individuals contributes to the systemic physiologic dysfunction of frailty.
PMCID: PMC2699396  PMID: 19194312
HIV; aging; frailty; CD4 T-cell count; Highly active antiretroviral therapy; prospective population-based cohort
21.  Regulatory T Cell Expansion and Immune Activation during Untreated HIV Type 1 Infection Are Associated with Disease Progression 
Regulatory T cells (Tregs) may play an important role in the immunopathology of chronic HIV-1 infection due to their potent suppressive activity of both T cell activation and effector function. To investigate the correlation between Tregs and immune activation during untreated chronic HIV-1 infection, we conducted a nested case–control study within the Multicenter AIDS Cohort Study (MACS). Twenty HIV-1-infected fast progressors (FP) and 40 slow progressors (SP) were included in our study using risk-set sampling. Nine age-matched HIV-1-uninfected men (UI) were also included. Cryopreserved peripheral blood mononuclear cells (PMBCs) were tested using flow cytometry analyses. We identified Tregs as Foxp3+CD25+CD4+ T cells and assessed the activation of CD4+ and CD8+ T cells by the expression of CD38, HLADR, or both markers simultaneously. There is a relative expansion of Tregs during HIV-1 infection, which is associated with disease progression. The increased CD38 expression on both CD4+ and CD8+ T cells expressed as either percentage or median fluorescence intensity (MFI) and the elevated proportion of CD8+ T cells that is HLADR+CD38+ were all associated with rapid HIV-1 progression. Counter to the assumed role of Tregs as the suppressors of activation, the expansion of Tregs was positively correlated with CD4+ T cell activation among HIV-1-infected fast progressors. The high level of Tregs associated with rapid HIV progression may suggest a detrimental role of these cells in the immune control of HIV-1 infection.
PMCID: PMC2782619  PMID: 19239357
22.  Regulatory T Cell Expansion and Immune Activation during Untreated HIV Type 1 Infection Are Associated with Disease Progression 
Regulatory T cells (Tregs) may play an important role in the immunopathology of chronic HIV-1 infection due to their potent suppressive activity of both T cell activation and effector function. To investigate the correlation between Tregs and immune activation during untreated chronic HIV-1 infection, we conducted a nested case–control study within the Multicenter AIDS Cohort Study (MACS). Twenty HIV-1-infected fast progressors (FP) and 40 slow progressors (SP) were included in our study using risk-set sampling. Nine age-matched HIV-1-uninfected men (UI) were also included. Cryopreserved peripheral blood mononuclear cells (PMBCs) were tested using flow cytometry analyses. We identified Tregs as Foxp3+CD25+CD4+ T cells and assessed the activation of CD4+ and CD8+ T cells by the expression of CD38, HLADR, or both markers simultaneously. There is a relative expansion of Tregs during HIV-1 infection, which is associated with disease progression. The increased CD38 expression on both CD4+ and CD8+ T cells expressed as either percentage or median fluorescence intensity (MFI) and the elevated proportion of CD8+ T cells that is HLADR+CD38+ were all associated with rapid HIV-1 progression. Counter to the assumed role of Tregs as the suppressors of activation, the expansion of Tregs was positively correlated with CD4+ T cell activation among HIV-1-infected fast progressors. The high level of Tregs associated with rapid HIV progression may suggest a detrimental role of these cells in the immune control of HIV-1 infection.
PMCID: PMC2782619  PMID: 19239357
23.  Telomerase-Based Pharmacologic Enhancement of Antiviral Function of Human CD8+ T Lymphocytes1 
Telomerase reverse transcribes telomere DNA onto the ends of linear chromosomes and retards cellular aging. In contrast to most normal somatic cells, which show little or no telomerase activity, immune cells up-regulate telomerase in concert with activation. Nevertheless, during aging and chronic HIV-1 infection, there are high proportions of dysfunctional CD8+ CTL with short telomeres, suggesting that telomerase is limiting. The present study shows that exposure of CD8+ T lymphocytes from HIV-infected human donors to a small molecule telomerase activator (TAT2) modestly retards telomere shortening, increases proliferative potential, and, importantly, enhances cytokine/chemokine production and antiviral activity. The enhanced antiviral effects were abrogated in the presence of a potent and specific telomerase inhibitor, suggesting that TAT2 acts primarily through telomerase activation. Our study is the first to use a pharmacological telomerase-based approach to enhance immune function, thus directly addressing the telomere loss immunopathologic facet of chronic viral infection.
PMCID: PMC2682219  PMID: 18981163
24.  Premature Aging of T cells Is Associated With Faster HIV-1 Disease Progression 
To determine if untreated HIV-1 infection and progression is associated with premature aging of memory CD8+ and CD4+ T cells and naive CD4+ T cells.
Twenty HIV-1–infected fast progressors and 40 slow progressors were included in our study, using risk set sampling. The expression of cell surface markers reflecting the differentiation stages of lymphocytes was measured using flow cytometry analyses performed on cryopreserved peripheral blood mononuclear cells.
We found that HIV-1 disease progression is associated with a decreased CD28 median florescence intensity on CD4+ and CD8+ T cells; an increased proportion of intermediate- and late-differentiated CD8+ T cells and a decreased CD31 median florescence intensity on naive CD4+ T cells of recent thymic origin. A selective depletion of peripherally expanded naive CD4+ T cells was found to be associated with HIV-1 infection but not with HIV-1 disease progression.
The overall change during HIV-1 infection and progression is associated with a shift in the T-cell population toward an aged conformation, which may be further compromised by impaired renewal of the less-differentiated CD4+ T-cell population. Our results suggest that HIV-1 infection induces an accelerated aging of T lymphocytes, which is associated with the clinical progression to AIDS and death.
PMCID: PMC2767229  PMID: 19131896
disease progression; HIV/AIDS; premature aging; T cells
25.  Emergence and Persistence of CXCR4-Tropic HIV-1 in a Population of Men from the Multicenter AIDS Cohort Study 
The Journal of infectious diseases  2008;198(8):1104-1112.
We examined the emergence of CXCR4 (i.e., X4) tropism in 67 male human immunodeficiency virus type 1 (HIV-1) seroconverters from the Multicenter AIDS Cohort Study (MACS) who were selected to reflect the full spectrum of rates of HIV-1 disease progression. A mean of 10 serial samples per donor were evaluated by a laboratory-validated, commercially available assay to determine phenotypic coreceptor use. A total of 52% of men had dual- or mixed-tropic HIV-1 detected at 1 or more of the time points tested. Use of X4 by HIV-1 was detected more frequently among men who developed AIDS (defined as a CD4+ T cell count of < 200 cells/μL and/or an AIDS-defining illness)≤11 years after seroconversion than among those who did not (P = .005), as well as among men who exhibited a total T cell count decline (i.e., a CD3+ inflection point), compared with those who did not (P = .03). For men in whom both X4 virus and an inflection point were detected, emergence of X4 virus preceded the inflection point by a median of 0.83 years. The median CD4+ T cell count at first detection of X4 viruses before the onset of AIDS was 475 cells/μL. We conclude that HIV-1 variants that used X4 frequently emerged at high CD4+ T cell counts and may contribute to the decrease in T cell numbers during late HIV-1 infection.
PMCID: PMC2753263  PMID: 18783316

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