Chronic infection by HIV increases the risk of cardiovascular disease (CVD) despite effective antiretroviral therapy (ART). The mechanisms linking HIV to CVD have yet to be fully elucidated. High plasma levels of the pro-inflammatory cytokine IL-6, which may be triggered by IL-1β, is a biomarker of CVD risk in HIV-negative adults, and of all-cause mortality in HIV disease. Monocytes play a pivotal role in atherosclerosis, and may be major mediators of HIV-associated inflammation. We therefore hypothesized that monocytes from HIV-infected adults would display high inflammatory responses. Employing a 10-color flow cytometry intracellular cytokine staining assay, we directly assessed cytokine and chemokine responses of monocytes from the cryopreserved peripheral blood of 33 chronically HIV-1 infected subjects. Participants were 45 years or older, on virologically suppressive ART and at risk for CVD. This group was compared to 14 HIV-negative subjects matched for age and gender, with similar CVD risk. We simultaneously detected intracellular expression of IL-1β, IL-6, IL-8 and TNF in blood monocytes in the basal state and after stimulation by triggers commonly found in the blood of treated, chronically HIV-infected subjects: lipopolysaccharide (LPS) and oxidized low-density lipoprotein (oxLDL). In the absence of stimulation, monocytes from treated HIV-infected subjects displayed a high frequency of cells producing IL-1β (median 19.5%), compared to low levels in HIV-uninfected persons (0.9% p<0.0001). IL-8, which is induced by IL-1β, was also highly expressed in the HIV-infected group in the absence of stimulation, 43.7% compared to 1.9% in HIV-uninfected subjects, p<0.0001. Strikingly, high basal expression of IL-1β by monocytes predicted high IL-6 levels in the plasma, and high monocyte IL-6 responses in HIV-infected subjects. Hyper-inflammatory IL-1β enriched monocytes may be a major source of IL-6 production and systemic inflammation in HIV-infected adults, and may contribute to the risk for all-cause mortality and cardiovascular disease in treated HIV infection.
Inflammation and hemostasis perturbation may be involved in vascular complications of HIV infection. We examined atherogenic biomarkers and subclinical atherosclerosis in HIV-infected adults before and after beginning highly-active antiretroviral therapy (HAART).
In the Women's Interagency HIV Study (WIHS), 127 HIV-infected women studied pre- and post-HAART were matched to HIV-uninfected controls. Six semi-annual measurements of soluble CD14, tumor necrosis factor (TNF)-alpha, soluble interleukin (IL)-2 receptor, IL-6, IL-10, monocyte chemoattractant protein (MCP)-1, D-dimer, and fibrinogen were obtained. Carotid artery intima-media thickness (CIMT) was measured by B-mode ultrasound.
Relative to HIV-uninfected controls, HAART-naïve HIV-infected women had elevated levels of soluble CD14 (1945 vs 1662 ng/mL, Wilcoxon signed rank P<0.0001), TNF-alpha (6.3 vs 3.4 pg/mL, P<0.0001), soluble IL-2 receptor (1587 vs 949 pg/mL, P<0.0001), IL-10 (3.3 vs 1.9 pg/mL, P<0.0001), MCP-1 (190 vs 163 pg/mL, P<0.0001) and D-dimer (0.43 vs 0.31 µg/mL, P<0.01). Elevated biomarker levels declined after HAART. While most biomarkers normalized to HIV-uninfected levels, in women on effective HAART, TNF-alpha levels remained elevated compared to HIV-uninfected women (+0.8 pg/mL, P=0.0002). Higher post-HAART levels of soluble IL-2 receptor (P=0.02), IL-6 (P=0.05), and D-dimer (P=0.03) were associated with increased CIMT.
Untreated HIV infection is associated with abnormal hemostasis (e.g., D-dimer), and pro-atherogenic (e.g., TNF-alpha) and anti-atherogenic (e.g., IL-10) inflammatory markers. HAART reduces most inflammatory mediators to HIV-uninfected levels. Increased inflammation and hemostasis are associated with subclinical atherosclerosis in recently treated women. These findings have potential implications for long-term risk of cardiovascular disease in HIV-infected patients, even with effective therapy.
antiretroviral therapy; cardiovascular diseases; cytokines; hemostasis; HIV; inflammation
From early in the HIV epidemic it was appreciated that many inflammatory markers such as neopterin and TNF-α were elevated in patients with AIDS. With the advent of modern technology able to measure a broad array of cytokines, we now know that from the earliest points of infection HIV induces a cytokine storm. This review will focus on how cytokines are disturbed in HIV infection and will explore potential therapeutic uses of cytokines. These factors can be used directly as therapy during HIV infection, either to suppress viral replication or prevent deleterious immune effects of infection, such as CD4+ T cell depletion. Cytokines also show great promise as adjuvants in the development of HIV vaccines, which would be critical for the eventual control of the epidemic.
HIV; cytokine; chemokine; immune activation; vaccine
Prior to the identification of hepatitis C virus (HCV), transfusion-transmission was common. Viral transmission in subjects with a known date of infection allows the study of the immune responses to acute HCV infection. We analysed 39 soluble immune factors in serum samples from subjects with transfusion-transmitted HCV. Dynamic expression kinetics of interferon gamma-induced protein 10 (IP-10), tumour necrosis factor-alpha and interleukin (IL)-10 were observed during acute HCV infection. Serum IP-10 was the only analyte that was significantly elevated in HCV resolvers compared with uninfected controls. In individuals who progressed to chronic HCV elevated levels of IP-10 and IL-10 coincided with first significant alanine aminotransferase elevation and remained elevated during the first year of acute HCV infection. In addition to monitoring lack of reduction in viral load, serum levels of IP-10 and IL-10 expression during acute HCV infection may be useful biomarkers to predict the progress to chronic HCV.
The RV144 vaccine trial in Thailand demonstrated that an HIV vaccine could prevent infection in humans and highlights the importance of understanding protective immunity against HIV. We used a nonhuman primate model to define immune and genetic mechanisms of protection against mucosal infection by the simian immunodeficiency virus (SIV). A plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen was evaluated for its ability to protect monkeys from infection by SIVmac251 or SIVsmE660 isolates after repeat intrarectal challenges. Although this prime-boost vaccine regimen failed to protect against SIVmac251 infection, 50% of vaccinated monkeys were protected from infection with SIVsmE660. Among SIVsmE660-infected animals, there was an about one-log reduction in peak plasma virus RNA in monkeys expressing the major histocompatibility complex class I allele Mamu-A*01, implicating cytotoxic T lymphocytes in the control of SIV replication once infection is established. Among Mamu-A*01–negative monkeys challenged with SIVsmE660, no CD8+ T cell response or innate immune response was associated with protection against virus acquisition. However, low levels of neutralizing antibodies and an envelope-specific CD4+ T cell response were associated with vaccine protection in these monkeys. Moreover, monkeys that expressed two TRIM5 alleles that restrict SIV replication were more likely to be protected from infection than monkeys that expressed at least one permissive TRIM5 allele. This study begins to elucidate the mechanism of vaccine protection against immunodeficiency viruses and highlights the need to analyze these immune and genetic correlates of protection in future trials of HIV vaccine strategies.
Recent-infection testing assays/algorithms (RITAs) have been developed to exploit the titer and avidity of HIV antibody evolution following seroconversion for incidence estimation. The Vitros Anti-HIV 1+2 assay (Ortho-Clinical Diagnostics) was approved by the FDA to detect HIV-1 and HIV-2 infections. We developed a less-sensitive (LS) and an avidity-modified version of this assay to detect recent HIV infection. Seroconversion panels (80 subjects, 416 samples) were tested to calculate the mean duration of recent infection (MDR) for these assays. A panel from known long-term (2+ years) HIV-infected subjects on highly active antiretroviral therapy (HAART) (n = 134) and subjects with low CD4 counts (AIDS patients [n = 140]) was used to measure the false-recent rate (FRR) of the assays. Using a signal-to-cutoff ratio of 20 and the LS-Vitros assay gave a RITA MDR of 215 days (95% confidence interval [95% CI], ±65 days) and using an avidity index (AI) of 0.6 gave an MDR of 170 days (±44 days), while a combination of the two assays yielded a MDR of 146 days (±38.6) and an FRR of 8%. Misclassifying subjects with known long-term infection as recently infected occurred in 14% of AIDS patients and 29% (95% CI, 22, 38) of HAART subjects and 3% (95% CI, 0.8, 7.2) and 42% (95% CI, 33, 51), respectively, for the LS- and avidity-modified Vitros assays, with a misclassification rate of 15% (95% CI, 11, 20) overall using a dual-assay algorithm. Both modified Vitros assays can be used to estimate the length of time since seroconversion and in calculations for HIV incidence. Like other RITAs, they are subject to high FRR in subjects on HAART or with AIDS.
Transfusion related acute lung injury (TRALI) has been associated with both HLA and HNA antibodies. HNA antibody frequency, specificity, and demographic associations have not been well defined in the blood donor population.
A subset of 1171 donors (388 non-transfused males, 390 HLA antibody negative females with three or more pregnancies, and 393 HLA antibody positive females with three or more pregnancies) from a larger leukocyte antibody prevalence study (LAPS) was tested for IgG and IgM HNA antibody using a granulocyte immunofluorescence flow cytometry assay. Additional testing on selected samples included monoclonal antibody immobilization of granulocyte antigen – flow cytometry and granulocyte genotyping.
Eight samples were HNA antibody positive (prevalence 0.7% [95% CI, 0.3 - 1.3%]). Three HNA antibodies (one IgG and two IgM) were found in non-transfused males (prevalence 0.8% [95% CI, 0.2 - 2.2%]); all were pan-reactive or non-specific. One HLA antibody negative previously pregnant female had an IgG HNA antibody with HNA-1a specificity (prevalence 0.3% [95% CI, 0.01-1.4%]). Four HLA antibody positive previously pregnant females demonstrated HNA antibodies, three IgG and one IgM (prevalence 1% [95% CI, 0.3 - 2.6%]). Two of these were HNA-1a specific, one HNA-4a specific, and one non-specific.
HNA antibodies occur with low frequency in the donor population and are present in both male and female donors. Despite the implementation of TRALI reduction strategies, HNA antibodies are still present in donor blood products. Though our data do not create a case for urgent implementation of donor HNA antibody testing, future new developments for high throughput HNA antibody screening, including for HNA-3a, may warrant reconsideration.
TRALI is the leading cause of transfusion-related deaths. Donor HLA antibodies have been implicated in TRALI cases. Blood centers are implementing TRALI risk reduction strategies based on HLA antibody screening of some subpopulations of ever-pregnant apheresis platelet donors. However, if screening assay cutoffs are too sensitive, donation loss may adversely impact blood availability.
Pregnancy history and HLA antibody screening and single antigen bead (SAB) data from blood donors in the REDS-II Leukocyte Antibody Prevalence Study (LAPS) were evaluated for correlations between assay screening values, HLA antibody titer, and number of HLA antigen specificities. The probabilities of matching a cognate antigen in a recipient were calculated and examined in association with total number of specificities observed and screening values. The relative impact of imposing various screening assay cutoffs or pregnancy stratification was examined in relation to detection of HLA antibody reactive donations and loss of donors and donations.
We provide evidence that higher HLA Ab screening assay values are associated with maintaining higher screening signals upon dilution and an increased breadth of specificities compared with lower screening values; the latter correlated with an increased risk of a cognate antigen match in potential recipients. Depending upon the TRALI risk reduction strategy used, the potential loss of donations ranged between 0.9 and 6.0%.
This analysis should enable blood centers to decide upon a TRALI risk reduction strategy for apheresis platelets that is consistent with how much donation loss the blood center can tolerate.
TRALI; Transfusion-Related Acute Lung Injury; HLA antibodies; platelet transfusions
HIV causes inflammation that can be at least partially corrected by HAART. To determine the qualitative and quantitative nature of cytokine perturbation, we compared cytokine patterns in three HIV clinical groups including HAART responders (HAART), untreated HIV non-controllers (NC), and HIV-uninfected (NEG).
Multiplex assays were used to measure 32 cytokines in a cross-sectional study of participants in the Women's Interagency HIV Study (WIHS). Participants from 3 groups were included: HAART (n=17), NC (n=14), and HIV NEG (n=17).
Several cytokines and chemokines showed significant differences between NC and NEG participants, including elevated IP-10 and TNF-α and decreased IL-12(p40), IL-15, and FGF-2 in NC participants. Biomarker levels among HAART women more closely resembled the NEG, with the exception of TNF-α and FGF-2. Secondary analyses of the combined HAART and NC groups revealed that IP-10 showed a strong, positive correlation with viral load and negative correlation with CD4+ T cell counts. The growth factors VEGF, EGF, and FGF-2 all showed a positive correlation with increased CD4+ T cell counts.
Untreated, progressive HIV infection was associated with decreased serum levels of cytokines important in T cell homeostasis (IL-15) and T cell phenotype determination (IL-12), and increased levels of innate inflammatory mediators such as IP-10 and TNF-α. HAART was associated with cytokine profiles that more closely resembled those of HIV uninfected women. The distinctive pattern of cytokine levels in the 3 study groups may provide insights into HIV pathogenesis, and responses to therapy.
HIV; CD4+ T cells; cytokines; chemokines; HAART
Background. Identifying persons with recent human immunodeficiency virus (HIV) antibody seroconversion is useful for treatment, research, and prevention, but the sensitivity and specificity of tests for this purpose are uncertain.
Methods. We used longitudinal specimens panels from 155 persons identified prior to HIV seroconversion to assess antibody-based methods for classifying persons as within 30, 60, or 90 days of seroconversion, including 2 incidence assays, a less-sensitive (LS) enzyme immunoassay (EIA), and the BED assay.
Results. Sensitivity and specificity, respectively, for identifying persons within 30 days of seroconversion were: 34%–57% and 98%–100% for 2 standard EIAs (employing a signal-to-cutoff ≤4.0; ≥1.0 defines HIV positive), 84% and 73% for the LS-EIA (≤0.2 cutoff), 88% and 72% for the BED (≤0.2 cutoff), and 43%–58% and 98% (≤3 bands) for 2 Western blot (WB) assays. By area under the receiver operator curves, the best test for identifying persons within 30 days of seroconversion was the number of bands on the Bio-Rad WB (0.90); within 60 days, the LS-EIA and BED (both 0.85); and for persons within 90 days the BED (0.86).
Conclusions. Standard EIAs, Western blots, and HIV incidence assays provide useful information for identifying persons 30 to 90 days after seroconversion.
Despite growing evidence that HIV-1-specific CD4+ T helper (Th) cells may play a role in the control of viremia, discrete Th cell epitopes remain poorly defined. Furthermore, it is not known whether Th cell responses generated using vaccines based on clade B virus sequences will elicit immune responses that are effective in regions of the world where non-clade B viruses predominate. To address these issues we isolated CD4+ T cell clones from individuals with vigorous HIV-1-specific Th cell responses and identified the minimum epitopes recognized. The minimum peptide length required for induction of CD4+ T cell proliferation, IFN-γ secretion, and cytolytic activity ranged from 9 to 16 amino acids in the five epitopes studied. Cross-clade recognition of the defined epitopes was examined for variant peptides from clades A, B, C, D, and AE. Over half the variant epitopes (17 of 32) exhibited impaired recognition, defined as less than 50% of the IFN-γ secretion elicited by B clade consensus sequence. There was no evidence for antagonistic activity mediated by the variant peptides, and despite strong responses there was no escape of autologous virus from Th responses in the epitopes we studied. Abrogated recognition of variant CD4+ T cell epitopes presents a potential obstacle to vaccine development.
It is thought that HLA antibodies might contribute to the pathogenesis of transfusion-related acute lung injury (TRALI). Many methods exist to detect HLA antibodies, including ELISA, flow cytometry, and multiplex bead-based assays, as well as the older lymphocytotoxicity assay. The sensitivity of each of these testing platforms varies, and it is not obvious how to compare HLA antibody results obtained on different platforms. This issue has become increasingly important as some blood centers have begun HLA antibody apheresis donor screening as a TRALI risk-reduction measure.
STUDY DESIGN AND METHODS
525 serum samples were selected from 7,841 donors in the Leukocyte Antibody Prevalence Study (LAPS) repository based on risk for the development of HLA antibodies, using the number of pregnancies as the risk factor. There were 81 males, and the pregnancy history of the 444 female donors was 0 (n=187), 1 (n=67), or 2+ pregnancies (n=190). Replicate frozen serum aliquots were sent to four different assay manufacturers blinded for HLA Ab detection using five different assays.
Among the assays with different manufacturer designated cutoffs, the flow cytometry and multiplex bead based-assays (Luminex) typically resulted in a larger proportion of HLA Ab positive samples compared with ELISA based assays. Capitalizing upon having quantitative results from five different assays on the same group of samples, latent variable analysis was used to derive a new set of cutoffs. These cutoffs, termed consensus cutoffs, yielded similar sensitivities across test platforms, thereby increasing concordance amongst assays. Assay agreement was higher in ever pregnant females than in males and never pregnant females.
Different assays resulted in varied positivity rates when the manufacturer’s suggested cutoffs were used, demonstrating that care needs to be taken when comparing clinical outcomes data generated using different HLA antibody assays and testing platforms. The method used here, involving latent variable analysis, presents one possible approach to calculating comparable cutoffs that result in broad agreement across assays with respect to positivity designation.
Nipah virus (NiV), a zoonotic paramyxovirus, is highly contagious in swine, and can cause fatal infections in humans following transmission from the swine host. The main viral targets in both species are the respiratory and central nervous systems, with viremia implicated as a mode of dissemination of NiV throughout the host. The presented work focused on the role of peripheral blood mononuclear cells (PBMC) in the viremic spread of the virus in the swine host. B lymphocytes, CD4−CD8−, as well as CD4+CD8− T lymphocytes were not permissive to NiV, and expansion of the CD4+CD8− cells early post infection was consistent with functional humoral response to NiV infection observed in swine. In contrast, significant drop in the CD4+CD8− T cell frequency was observed in piglets which succumbed to the experimental infection, supporting the hypothesis that antibody development is the critical component of the protective immune response. Productive viral replication was detected in monocytes, CD6+CD8+ T lymphocytes and NK cells by recovery of infectious virus in the cell supernatants. Virus replication was supported by detection of the structural N and the non-structural C proteins or by detection of genomic RNA increase in the infected cells. Infection of T cells carrying CD6 marker, a strong ligand for the activated leukocyte cell adhesion molecule ALCAM (CD166) highly expressed on the microvascular endothelial cell of the blood-air and the blood-brain barrier may explain NiV preferential tropism for small blood vessels of the lung and brain.
Highly active antiretroviral therapy (HAART) can effectively reduce plasma HIV RNA levels to below the level of detection in most HIV-infected patients. The degree to which residual low-level viremia persists during HAART remains unclear.
We identified 180 subjects (median duration of HIV infection 12 years) who had ≥2 consecutive plasma HIV-1 RNA levels below the level of detection (<50-75 copies/mL) while taking antiretroviral drugs; 36/180 had been virologically suppressed for >5 years. Longitudinal plasma samples that were taken from these subjects during periods of viral load suppression were selected and analyzed. The isothermal Transcription Mediated Amplification (TMA) (limit of detection <3.5 copies RNA/mL) assay was used to measure persistent viremia. A “detuned” EIA assay was used to obtain quantitative HIV antibody levels.
A total of 1606 TMA assays were performed on 438 specimens in 180 HAART-suppressed subjects (median 3 replicates per specimen). In the first year of viral suppression, plasma RNA levels declined significantly (p=0.001), but after month 12 there was no evidence for a continued decline (p=0.383). In the first year of viral suppression, HIV antibody levels also declined (p=0.054), but after month 12 there was no evidence for a continued decline (p=0.988).
Viremia continued to decline during the first 12 months after viremia became undetectable using conventional methods, and then remained stable. HIV antibody levels also decreased in the first year of viral suppression and then remained stable. Viremia and the HIV-associated host response appear to achieve a steady-state “set-point” during long-term combination therapy.
Residual viremia; HAART-suppressed; HIV antibody levels
Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality. Blood centers are implementing TRALI risk reduction strategies based on screening apheresis donors for antibodies to human leukocyte antigens (HLA).
Study Design and Methods
HLA antibody screening was performed on 7920 blood donors from the Leukocyte Antibody Prevalence Study (LAPS) using Luminex based Normalized Background (NBG) cutoff ratios of 10.8 (class I) and 6.9 (class II). Single antigen bead (SAB) assay cutoffs of 2500 Median Fluorescence Intensity (MFI) units (class I) and 1500 (class II) were established based on results of two subpopulations of LAPS donors. Antibody frequencies against HLA A, B, C, DR, DQ and DP antigens were determined for screen-reactive donors with prior pregnancies.
SAB reactivity for samples above our multi-antigen bead NBG cutoffs was 78% for class I and 79% for class II. The SAB positive rate increased among women with zero to ≥4 pregnancies (0.3% - 15.6% class I and 0.4% - 18% class II; p<0.00001). The highest frequency antibodies were DR11 and B15 (4.4% of women with prior pregnancies). The majority of class I positives contained >5 specificities. For class II, antibody positive women segregated into two groups; a single specificity or >5 specificities.
Identification of HLA antigen specificities supports pregnancy associations previously found with screening assays. The significance of particular HLA specificities for inducing TRALI is currently being evaluated in a large lookback study of recipients of high plasma volume components from this donor cohort.
TRALI; HLA antibody; blood donors; LAPS
Measurement of peripheral blood cytokines and other immunomodulatory proteins is a useful and popular tool for assessing human immune responses to a wide range of assaults. A common challenge in this work is obtaining fresh, high-quality samples and limiting the time between blood collection and the separation of plasma or serum from cells. In this study we sought to determine the effect of sample age at the time of processing on the measured levels of 41 soluble immune mediators. Two cohorts were examined: healthy lab donors and trauma patients, who have significant immune perturbation. Whole-blood samples were aliquoted, and plasma was isolated, at days 0, 1, 2, and 3 after collection. Multiplexing techniques were used to measure protein concentrations, and general estimating equations were used to determine if there was a significant change over time. Over the 3-day period examined, only 15 of the 41 proteins showed no significant change in either cohort. Among the remaining proteins both increases and decreases were observed, with changes ranging from 2.4% per day to 325% per day. Proteins with significant changes in one cohort did not always show significant changes in the other group. These results support the need to separate plasma or serum from whole blood as quickly as possible and/or to standardize the length of time to processing within a given study of peripheral blood protein concentrations. When this is not possible, care should be taken to account for differences due to sample age.
HIV+ elite controllers are a unique group of rare individuals who maintain undetectable viral loads in the absence of antiretroviral therapy. We studied immune responses in these subjects to inform vaccine development, with the goal of identifying the immune correlates of protection from HIV.
We compared markers of cellular activation, HIV-specific immune responses, and regulatory T (Treg) cell frequencies in 4 groups of subjects: HIV-negative healthy controls, elite controllers (HIV RNA level <75 copies/ml), individuals on highly active antiretroviral therapy (HAART), and subjects with HIV RNA level >10,000 copies/ml (non-controllers).
Elite controllers possessed significantly lower levels of activated HIV-specific CD8+ T cells and of recently divided HIV-specific CD4+ T cells than non-controllers, while these differences were not seen in the respective CMV-specific T cell populations. Elite controllers also mounted a stronger and broader cytokine and chemokine response following HIV-specific stimulation than individuals on HAART and non-controllers. Finally, we found that HAART suppressed subjects had elevated Treg cell frequencies, while elite controllers and non-controllers maintained normal percentages of Treg cells.
Elite controllers maintain high levels of HIV-specific immune responses with low levels of HIV-specific T cell activation, and do not have elevated Treg cell levels. Based on these data an ideal HIV vaccine would induce strong HIV-specific immune responses while minimizing HIV-specific T cell activation.
Cellular immunity; activation; regulatory T cells; pathogenesis; HIV
Acute HIV infection (AHI) is a critical phase of infection when irreparable damage to the immune system occurs and subjects are very infectious. We studied subjects with AHI prospectively to develop better treatment and public health interventions.
Cross-sectional screening was employed to detect HIV RNA positive, antibody negative subjects. Date of HIV acquisition was estimated from clinical history and correlated with sequence diversity assessed by single genome amplification (SGA). Twenty-two cytokines/chemokines were measured from enrollment through week 24.
Thirty-seven AHI subjects were studied. In 7 participants with limited exposure windows, the median exposure to HIV occurred 14 days before symptom onset. Lack of viral sequence diversification confirmed the short duration of infection. Transmission dates estimated by SGA/sequencing using molecular clock models correlated with transmission dates estimated by symptom onset in individuals infected with single HIV variants (mean of 28 versus 33 days). Only 10 of 22 cytokines/chemokines were significantly elevated among AHI participants at enrollment compared to uninfected controls, and only 4 participants remained seronegative at enrollment.
The results emphasize the difficulty in recruiting subjects early in AHI. Viral sequence diversity proved accurate in estimating time of infection. Regardless of aggressive screening, peak viremia and inflammation occurred before enrollment and potential intervention. Given the personal and public health importance, improved AHI detection is urgently needed.
HLA antibodies are a possible cause of transfusion-related acute lung injury (TRALI), and fluorescent bead assays are often used for antibody detection. Serum is the manufacturer’s recommended sample, but plasma may be easier to obtain for studies of HLA antibody prevalence and TRALI case investigations.
STUDY DESIGN AND METHODS
Specimens were obtained from 44 multiparous females positive for HLA antibodies by lymphocytotoxicity testing at least 13 years prior, and from 1,000 contemporary blood donors. Screening tests were performed using a Luminex-based assay. In addition to comparing results obtained with paired plasma and serum samples, the effects of storage at 4 °C for one week and of multiple freeze-thaw cycles were evaluated.
Of 42 evaluable subjects with HLA antibodies documented >13 years earlier, only 1 showed loss of detectable antibodies, with 39 (93%) positive in the screening assay for class I and 24 (57%) positive in the screening assay for HLA class II antibodies. In 968 evaluable contemporary donors, 291 screened positive for HLA class I and 206 for HLA class II antibodies using a low assay cut-off. Screening test concordance using paired plasma and serum samples was high, particularly for subjects with higher level antibodies. Refrigeration of samples for one week did not significantly affect assay results, while repeated freeze-thaw cycles caused a decrement in signal level.
Serum and plasma samples gave concordant results in the majority of cases, particularly for specimens with higher-level antibodies. High-level HLA antibodies were present in most individuals for over 13 years.
HIV-infected individuals maintaining undetectable viremia in the absence of therapy (HIV controllers) often maintain high HIV-specific T cell responses, which has spurred the development of vaccines eliciting HIV-specific T cell responses. However, controllers also often have abnormally high T cell activation levels, potentially contributing to T cell dysfunction, CD4+ T cell depletion, and non-AIDS morbidity. We hypothesized that a weak T regulatory cell (Treg) response might contribute to the control of viral replication in HIV controllers, but might also contribute to generalized immune activation, contributing to CD4+ T cell loss. To address these hypotheses, we measured frequencies of activated (CD38+ HLA-DR+), regulatory (CD4+CD25+CD127dim), HIV-specific, and CMV-specific T cells among HIV controllers and 3 control populations: HIV-infected individuals with treatment-mediated viral suppression (ART-suppressed), untreated HIV-infected “non-controllers” with high levels of viremia, and HIV-uninfected individuals. Despite abnormally high T cell activation levels, controllers had lower Treg frequencies than HIV-uninfected controls (P = 0.014). Supporting the propensity for an unusually low Treg response to viral infection in HIV controllers, we observed unusually high CMV-specific CD4+ T cell frequencies and a strong correlation between HIV-specific CD4+ T cell responses and generalized CD8+ T cell activation levels in HIV controllers (P≤0.001). These data support a model in which low frequencies of Tregs in HIV controllers may contribute to an effective adaptive immune response, but may also contribute to generalized immune activation, potentially contributing to CD4 depletion.
The complexity of immunoregulation has focused attention on the CD4+ T “suppressor” regulatory cell (Treg), which helps maintain balance between immunity and tolerance. An immunoregulatory T cell population that upon activation amplifies cellular immune responses was described in murine models more than thirty years ago. However, no study has yet identified a naturally occurring T “inducer” cell type. Here, we report that the ectoenzyme CD39/NTPDase1 (ecto-nucleoside triphosphate diphosphohydrolase 1) helps to delineate a novel population of human “inducer” CD4+ T cells (TInd) that significantly increases the proliferation and cytokine production of responder T cells in a dose-dependent manner. Furthermore, this unique TInd cell subset produces a distinct repertoire of cytokines in comparison to the other CD4+ T cell subsets. We propose that this novel CD4+ T cell population counterbalances the suppressive activity of suppressor Treg cells in peripheral blood and serves as a calibrator of immunoregulation.
CD4; CD39; Regulatory T cells; FOXP3; CD127; CD25; proliferation; Inducer; cytokine; IFN-γ; IL-6; IL-10; TNF-α; GM-CSF; ATP
Most human T cell leukemia virus type 1 (HTLV-1) infected subjects remain asymptomatic throughout their lives, with a few individuals developing HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukemia. Lymphocytes from about half of HTLV-1 infected subjects spontaneously proliferate in vitro, and how this phenomenon relates to symptomatic disease outcome and viral burden is poorly understood. Spontaneous proliferation was measured in lymphocyte subsets, and these findings were correlated with HTLV-1 proviral load and Tax expression in PBMCs. We found that in addition to previously described vigorous CD8+ T cell spontaneous proliferation, natural killer (NK) cells spontaneously proliferated to a similar high level, resulting in expansion of CD56-expressing NK cells. Spontaneous NK cell proliferation positively correlated with HTLV-1 proviral load but not with Tax expression or the presence of HAM/TSP. The strongest correlate with clinical outcome in this cohort was the ability of cells to express Tax, while HTLV-1 proviral load was more closely related to spontaneous NK cell proliferation. These results demonstrate that spontaneous proliferation, Tax expression, and proviral load are inter-related but not equivalent, and that spontaneous lymphocyte proliferation is not restricted to T cells, the targets of HTLV-1 infection.
Most human T cell leukemia virus type 1 (HTLV-1) infected subjects remain asymptomatic throughout their lives, with a few individuals developing HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukemia. Lymphocytes from about half of HTLV-1 infected subjects spontaneously proliferate in vitro, and how this phenomenon relates to symptomatic disease outcome and viral burden is poorly understood. Spontaneous proliferation was measured in lymphocyte subsets, and these findings were correlated with HTLV-1 proviral load and Tax expression in peripheral blood mononuclear cells (PBMCs). We found that in addition to previously described vigorous CD8+ T cell spontaneous proliferation, natural killer (NK) cells spontaneously proliferated to a similar high level, resulting in expansion of CD56-expressing NK cells. Spontaneous NK cell proliferation positively correlated with HTLV-1 proviral load but not with Tax expression or the presence of HAM/TSP. The strongest correlate with clinical outcome in this cohort was the ability of cells to express Tax, while HTLV-1 proviral load was more closely related to spontaneous NK cell proliferation. These results demonstrate that spontaneous proliferation, Tax expression, and proviral load are inter-related but not equivalent, and that spontaneous lymphocyte proliferation is not restricted to T cells, the targets of HTLV-1 infection.
HTLV; T cell; NK cell; Tax; HAM/TSP