High CD8+ T cell activation marks a less differentiated HIV-1 specific CD8+ T cell response that is not altered by suppression of viral replication.

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

Poor predictive value of cytomegalovirus (CMV)-specific T cell assays for the development of CMV retinitis in patients with AIDS.

BACKGROUND: We examined the potential clinical utility of using a cytomegalovirus (CMV)-specific T cell immunoassay to determine the risk of developing new-onset CMV retinitis (CMVR) in patients with acquired immunodeficiency syndrome (AIDS). METHODS: CMV-specific T cell assays were performed by multiparameter flow cytometry using stored peripheral blood mononuclear cells that had been obtained in an observational study 2-6 months before new-onset CMVR was diagnosed in case patients (at a study visit during which a dilated ophthalmologic examination revealed no evidence of CMVR) and at the same study visit in control subjects (matched by absolute CD4(+) T cell count at entry) who did not subsequently develop retinitis during 1-6 years of study follow-up. RESULTS: There were no significant differences in CMV-specific CD4(+) or CD8(+) T cell interferon-gamma or interleukin-2 expression in peripheral blood mononuclear cells from case patients and control subjects. Although there were trends toward lower percentages and absolute numbers of CMV-specific, cytokine-expressing CD8(+) T cells with a "late memory" phenotype (CD27(-)CD28(-)) as well as with an "early memory" phenotype (CD27(+)CD28(+)CD45RA(+)) in case patients than in control subjects, these differences were not statistically significant. CONCLUSIONS: Many studies have reported that CMV-specific CD4(+) and CD8(+) T cell responses distinguish patients with active CMVR (i.e., who lack CMV-protective immunity) from those with inactive CMVR after immune restoration by antiretroviral treatment (i.e., who have CMV-protective immunity). However, the multiple CMV-specific immune responses we measured do not appear to have clinical utility for predicting the risk for patients with AIDS of developing new-onset CMVR with sufficient accuracy to be used in guiding therapeutic management.

Increased carotid intima-media thickness in HIV patients is associated with increased cytomegalovirus-specific T-cell responses.

OBJECTIVES: HIV-infected subjects are at increased risk for myocardial infarction. The mechanism of this increased risk remains unclear. Since cytomegalovirus (CMV) infection has been associated with accelerated atherosclerosis in the transplant population and immune responses against CMV may be altered by HIV disease, we hypothesized that enhanced T-cell responses against CMV would be associated with increased atherosclerosis in subjects with HIV. METHODS: We measured high-sensitivity C-reactive protein (hs-CRP), T-cell activation, CMV-specific T-cell responses, and carotid artery intima-media thickness (IMT) in 93 HIV-infected subjects and in 37 uninfected controls. RESULTS: The mean age of the HIV-infected subjects was 48 years and 85 (91%) were male. The median carotid IMT was higher in the HIV-infected group compared to the uninfected group (0.95 mm versus 0.68 mm, P < 0.001). This difference remained significant after controlling for all traditional risk factors. Compared to HIV-negative controls, HIV-infected subjects had higher median levels of hs-CRP (P = 0.05), higher levels of CD4 and CD8 T-cell activation (P < 0.0001) and higher CMV-specific interferon-gamma CD8 T-cell responses (P < 0.0001). CMV-specific T-cell responses, but not hs-CRP and T-cell activation, were independently associated with higher carotid IMT (P = 0.001). CONCLUSIONS: HIV-infected subjects had thicker carotid IMT compared to controls. While HIV patients also had higher T-cell activation, hs-CRP levels, and CMV-specific T-cell responses, only CMV-specific T-cell responses were independently associated with IMT. Accelerated atherosclerosis in HIV patients may be mediated by heightened CMV-induced immune responses.

Protective immunity to cytomegalovirus (CMV) retinitis in AIDS is associated with CMV-specific T cells that express interferon- gamma and interleukin-2 and have a CD8+ cell early maturational phenotype.

To determine potential correlates of immune recovery from AIDS-related cytomegalovirus retinitis (CMVR), multiparameter flow cytometry was used to characterize CMV-specific T cells from subjects with CMVR. Individuals with active retinitis were compared with those who had been clinically immunorestored by antiretroviral therapy and had > or =2 years of ophthalmologic follow-up without anti-CMV therapy or retinitis reactivation or progression. In comparison with patients with active retinitis, immunorestored patients had higher circulating CD4(+) and CD8(+) T cells expressing interleukin-2 and interferon- gamma in response to combined CMV pp65 and IE1 peptide pool stimulation. CD4(+) T cell responses were predominantly to pp65, whereas CD8(+) T cell responses were predominantly to IE. Immunorestored patients, compared with patients with active retinitis, had increased levels of circulating CMV-specific CD8(+) T cells with "early" (CD27(+)CD28(+)CD45RA(+), CD27(+)CD28(+)CD45RA(-)) and "intermediate" (CD27(-)CD28(+)CD45RA(-)) phenotypes. Recovery from AIDS-related CMVR after the initiation of antiretroviral therapy may be mediated by CMV-specific CD4(+) and CD8(+) T cells capable of promoting antigen-specific CD8(+) T cell proliferation.

Safety and immunogenicity of Towne cytomegalovirus vaccine with or without adjuvant recombinant interleukin-12.

The Towne, human cytomegalovirus (CMV) vaccine is safe and immunogenic but has not prevented infection at doses tested to date. We administered 3000 pfu Towne CMV vaccine, with or without adjuvant recombinant interleukin-12 (rhIL-12), to CMV-seronegative healthy volunteers and then measured CMV gB-specific IgG titers and CMV-specific CD4+ and CD8+ T cell proliferation and IFNgamma expression after stimulation with whole viral lysate and immunodominant peptide CMV antigens. Adjuvant rhIL-12 at doses up to 2 microg were well-tolerated and associated with (1) dose-related increases in peak anti-CMV gB IgG titers (though not in sustained titers), (2) dose-related increases in the weak CMV viral lysate-specific CD4+ T cell proliferation responses induced by vaccine alone after 360 days of follow-up, and (3) decreases in the very robust CMV IE-specific peak CD4+ T cell and Day 360 CD8+ T cell proliferation responses induced by the vaccine alone. Also, qualitative CD8+ T cell IFNgamma responses to stimulation with the immunodominant CMV antigen, pp65, tended to occur more frequently in vaccinees who received 0.5-2.0 microg rhIL-12 compared to lower dose or no rhIL-12. Thus, adjuvant IL-12 may be a promising strategy for improving antibody and T cell immune responses to a CMV vaccine.

Antigen-specific T cell responses induced by Towne cytomegalovirus (CMV) vaccine in CMV-seronegative vaccine recipients.

BACKGROUND: Towne cytomegalovirus (CMV) vaccine is safe and immunogenic, though its protective efficacy has yet to be optimized. OBJECTIVE: Describe antigen-specific T cell responses to Towne vaccination. STUDY DESIGN: 3000 pfu Towne CMV vaccine were given to 12 CMV-seronegative volunteers. CMV-specific CD4+ and CD8+ T cell proliferation and IFNgamma expression were measured by flow cytometry after stimulation with CMV lysate or peptides. RESULTS: All vaccinees developed CD4+ and CD8+ T cell proliferation and CD4+ T cell IFNgamma responses to multiple CMV antigens, but their CD8+ T cells had low or undetectable IFNgamma responses to pp65 peptide pool. The seven HLA-A2+ subjects had higher CD8+ T cell proliferation and IFNgamma responses to IE than pp65, and two never developed CD8+ T cell IFNgamma responses to pp65. Peak CD4+ T cell IFNgamma responses to CMV lysate were lower than values observed in natural CMV seropositives. Initial CD4+ and CD8+ T cell responses to lysate and pp65 waned after 12 months to levels that were lower than those in healthy CMV seropositives, while vaccinees' CD8+ T cell responses to IE were robust and prolonged. CONCLUSION: Correlating CMV antigen-specific T cell responses with clinical protective efficacy may facilitate future CMV vaccine development.

Standardization of cytokine flow cytometry assays.

BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

T-cell activation and memory phenotypes in cerebrospinal fluid during HIV infection.

We characterized T cell phenotypes in 74 paired blood and cerebrospinal fluid (CSF) samples of HIV-infected and uninfected persons using four-color flow cytometry. CD4+ and CD8+ T cells subsets were further characterized by identifying activated/resting and memory/naive subsets in CSF and blood using the markers CD38/HLA-DR and CD45RA/CD62L, respectively. With and without HIV-infection, the proportion of CD4+ T cells and memory T cells among T cells in CSF was higher compared to blood. In HIV-infection, activated CD4+ and CD8+ T cells in CSF were more abundant than in uninfected controls. As expected, combination antiretroviral therapy (ART) reduced T cell activation in CSF and blood.

CMV antigen-specific CD4+ and CD8+ T cell IFNgamma expression and proliferation responses in healthy CMV-seropositive individuals.

CMV-specific CD4+ and CD8+ T cell IFNgamma expression and proliferation were measured in healthy volunteers by flow cytometry after CMV lysate or CMV pp65 or IE peptide pool stimulation. Cutoff values were set to maximize specificity (i.e., no false positive CMV-seronegatives). Sensitivity (defined as a positive response in CMV-seropositives to at least one of the 3 antigen preparations used) was 100% for CMV-specific CD4+ and CD8+ T cell IFN expression and CD4+ T cell proliferation and 95.4% for CMV-specific CD8+ T cell proliferation. All 22 CMV-seropositive individuals had positive responses by at least three of these four measurements. These findings support the concept that a multiplicity of antigen-specific functional immune responses and persistence of robust virus-specific CD4+T cells are important components of protective immunity in this chronic viral infection.

Results of a cytomegalovirus (CMV)-specific CD8+/interferon- gamma+ cytokine flow cytometry assay correlate with clinical evidence of protective immunity in patients with AIDS with CMV retinitis.

To evaluate the potential clinical utility of a cytomegalovirus (CMV)-specific CD8+/interferon (IFN)- gamma+ cytokine flow cytometry (CFC) assay for patients with CMV retinitis (CMVR), stored peripheral blood mononuclear cell specimens were obtained from patients with active CMVR (i.e., having clinical evidence of absent CMV-protective immunity), as well as from highly active antiretroviral therapy-treated patients with CMVR who were able to discontinue anti-CMV therapy without subsequent progression of retinitis (i.e., having clinical evidence of restored CMV-protective immunity). Positive CD8+/IFN- gamma+ T lymphocyte responses to CMV phosphoprotein 65 or immediate early peptide-pool stimulation were present in specimens from only 3 of 10 patients with active CMVR but were present in at least 1 specimen from all 20 patients with immunorestored CMVR, with a mean of 2.4 specimens/patient tested, spanning up to 6 months of observation (P = .0001). Among the patients with immunorestored CMVR, positive responses were present in all longitudinal specimens from 15 of the 20 patients. These data suggest that further testing of the CMV-specific CD8+/IFN- gamma+ CFC assay, for clinical utility in predicting incident and progressive CMVR disease, is warranted.

Strong cell-mediated immune responses are associated with the maintenance of low-level viremia in antiretroviral-treated individuals with drug-resistant human immunodeficiency virus type 1.

Antiretroviral (ARV)-treated patients often maintain low to moderate levels of viremia, despite the emergence of drug-resistant human immunodeficiency virus (HIV). We studied host and viral factors that may contribute to the control of viral replication in a cohort of 189 adults. Among ARV-treated patients with detectable viremia, there was a bell-shaped relationship between Gag-specific CD4+ T cell responses and viremia, with the highest cellular immune responses observed in patients with plasma HIV RNA levels of 1000-10,000 copies/mL. In contrast, there was a negative association between Gag-specific CD4+ T cell responses and viremia among ARV-untreated individuals with wild-type HIV. Strong cellular immune responses among individuals with drug-resistant HIV predicted subsequent lack of virological progression. Finally, there was a positive correlation between replicative capacity and viremia. Collectively, these data suggest that the selection of drug-resistance mutations may reduce the pathogenic potential of HIV, which leads to a balanced state of enhanced cellular immunity and low-level viremia.

Comparison of the ELISPOT and cytokine flow cytometry assays for the enumeration of antigen-specific T cells.

The enumeration of antigen-specific T cell responses has been greatly facilitated in recent years by the development of methods based on the detection of cytokines. In particular, the enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) assays have become popular. Since both assays are likely to continue to be in widespread use, it is important to evaluate whether their results are comparable. In the current study, we compared the results obtained in the ELISPOT and CFC assays using peptide pools corresponding to CMV and HIV-1 proteins in chronically HIV-1-infected individuals. Analysis of T cell responses to peptide pools indicated that the CMV pp65 and HIV-1 Gag CFC and ELISPOT-derived results were statistically correlated. However, the results obtained with each assay differed in important ways: the magnitude of the response was consistently higher in the CFC assay while the CFC assay was less likely than the ELISPOT assay to detect low-level responses. Furthermore, there was a lack of numeric agreement between ELISPOT and CFC results. For studies that require the detection of low-level responses, or definition of responses as positive or negative, the ELISPOT assay may be preferable. In contrast, the CFC has a greater dynamic range and allows for phenotypic discrimination of responding cells, making it the assay of choice for most other applications.

Short-term effects of cannabinoids in patients with HIV-1 infection: a randomized, placebo-controlled clinical trial.

BACKGROUND: Cannabinoid use could potentially alter HIV RNA levels by two mechanisms: immune modulation or cannabinoid-protease inhibitor interactions (because both share cytochrome P-450 metabolic pathways). OBJECTIVE: To determine the short-term effects of smoked marijuana on the viral load in HIV-infected patients. DESIGN: Randomized, placebo-controlled, 21-day intervention trial. SETTING: The inpatient General Clinical Research Center at the San Francisco General Hospital, San Francisco, California. PARTICIPANTS: 67 patients with HIV-1 infection. INTERVENTION: Participants were randomly assigned to a 3.95%-tetrahydrocannabinol marijuana cigarette, a 2.5-mg dronabinol (delta-9-tetrahydrocannabinol) capsule, or a placebo capsule three times daily before meals. MEASUREMENTS: HIV RNA levels, CD4+ and CD8+ cell subsets, and pharmacokinetic analyses of the protease inhibitors. RESULTS: 62 study participants were eligible for the primary end point (marijuana group, 20 patients; dronabinol group, 22 patients; and placebo group, 20 patients). Baseline HIV RNA level was less than 50 copies/mL for 36 participants (58%), and the median CD4+ cell count was 340 x 109 cells/L. When adjusted for baseline variables, the estimated average effect versus placebo on change in log10 viral load from baseline to day 21 was -0.07 (95% CI, -0.30 to 0.13) for marijuana and -0.04 (CI, -0.20 to 0.14) for dronabinol. The adjusted average changes in viral load in marijuana and dronabinol relative to placebo were -15% (CI, -50% to 34%) and -8% (CI, -37% to 37%), respectively. Neither CD4+ nor CD8+ cell counts appeared to be adversely affected by the cannabinoids. CONCLUSIONS: Smoked and oral cannabinoids did not seem to be unsafe in people with HIV infection with respect to HIV RNA levels, CD4+ and CD8+ cell counts, or protease inhibitor levels over a 21-day treatment.

Performance of plate-based cytokine flow cytometry with automated data analysis.

BACKGROUND: Cytokine flow cytometry (CFC) provides a multiparameter alternative to ELISPOT assays for rapid quantitation of antigen-specific T cells. To increase the throughput of CFC assays, we have optimized methods for stimulating, staining, and acquiring whole blood or PBMC samples in 96-well or 24-well plates. RESULTS: We have developed a protocol for whole blood stimulation and processing in deep-well 24- or 96-well plates, and fresh or cryopreserved peripheral blood mononuclear cell (PBMC) stimulation and processing in conventional 96-well round-bottom plates. Samples from both HIV-1-seronegative and HIV-1-seropositive donors were tested. We show that the percent response, staining intensity, and cell recovery are comparable to stimulation and processing in tubes using traditional methods. We also show the equivalence of automated gating templates to manual gating for CFC data analysis. CONCLUSION: When combined with flow cytometry analysis using an automated plate loader and an automated analysis algorithm, these plate-based methods provide a higher throughput platform for CFC, as well as reducing operator-induced variability. These factors will be important for processing the numbers of samples required in large clinical trials, and for epitope mapping of patient responses.

T cell activation is associated with lower CD4+ T cell gains in human immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral therapy.

Although T cell activation is associated with disease progression in untreated human immunodeficiency virus type 1 (HIV-1) infection, its significance in antiretroviral-treated patients is unknown. Activated (CD38(+)HLA-DR(+)) T cell counts were measured in 99 HIV-infected adults who had maintained a plasma HIV RNA level

Short-term effects of cannabinoids on immune phenotype and function in HIV-1-infected patients.

Cannabinoids, including smoked marijuana and delta9-tetrahydrocannabinol (THC) (dronabinol, Marinol), have been used to treat human immunodeficiency virus-1 (HIV)-associated anorexia and weight loss. Concerns have been raised, however, that these compounds might have adverse effects on the immune system of subjects with HIV infection. To determine whether such effects occur, the authors designed a randomized, prospective, controlled trial comparing the use of marijuana cigarettes (3.95% THC), dronabinol (2.5 mg), and oral placebo in HIV-infected adults taking protease inhibitor-containing highly active antiretroviral therapy (HAART). Assays of immune phenotype (including flow cytometric quantitation of T cell subpopulations, B cells, and natural killer [NK] cells) and immunefunction (including assays for induced cytokine production, NK cell function, and lymphoproliferation) were performed at baseline and weekly thereafter. On the basis of these measurements and during this short 21-day study period, few statistically significant effects were noted on immune system phenotypes orfunctions in this patient population.

A pilot study of the discontinuation of antifungal therapy for disseminated cryptococcal disease in patients with acquired immunodeficiency syndrome, following immunologic response to antiretroviral therapy.

To determine whether microbiologic cure of acquired immunodeficiency syndrome (AIDS)-related disseminated cryptococcosis is possible in patients receiving highly active antiretroviral therapy (HAART), antifungal therapy was discontinued in 6 patients with a history of disseminated cryptococcosis who had received > or =12 months of antifungal therapy. All were asymptomatic and had absolute CD4+ T cell counts of >150 cells/microL (range, 178-525 cells/microL). Blood, cerebrospinal fluid (CSF), and urine samples were obtained for fungal culture. Serum and CSF cryptococcal antigen titers were also obtained. All 6 patients had CSF and blood cultures negative for Cryptococcus neoformans and were receiving HAART. All patients' subsequent cultures remained sterile, and all patients were clinically asymptomatic 24 months after ending antifungal therapy. Disseminated cryptococcal disease can be cured by prolonged antifungal therapy in some patients with AIDS who experience sustained CD4 lymphocyte increases while receiving HAART.