Poxvirus Antigen Staining of Immune Cells as a Biomarker to Predict Disease Outcome in Monkeypox and Cowpox Virus Infection in Non-Human Primates

Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1–2×10⁷ PFU) or CPXV (>10² PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection.     

Infrared Spectroscopic Studies of Cells and Tissues: Triple Helix Proteins as a Potential Biomarker for Tumors

In this work, the infrared (IR) spectra of living neural cells in suspension, native brain tissue, and native brain tumor tissue were investigated. Methods were developed to overcome the strong IR signal of liquid water so that the signal from the cellular biochemicals could be seen. Measurements could be performed during surgeries, within minutes after resection.Comparison between normal tissue, different cell lineages in suspension, and tumors allowed preliminary assignments of IR bands to be made. The most dramatic difference between tissues and cells was found to be in weaker IR absorbances usually assigned to the triple helix of collagens. Triple helix domains are common in larger structural proteins, and are typically found in the extracellular matrix (ECM) of tissues.An algorithm to correct offsets and calculate the band heights and positions of these bands was developed, so the variance between identical measurements could be assessed. The initial results indicate the triple helix signal is surprisingly consistent between different individuals, and is altered in tumor tissues. Taken together, these preliminary investigations indicate this triple helix signal may be a reliable biomarker for a tumor-like microenvironment. Thus, this signal has potential to aid in the intra-operational delineation of brain tumor borders.     

Analysis of Mycobacterium tuberculosis-Specific CD8 T-Cells in Patients with Active Tuberculosis and in Individuals with Latent Infection

CD8 T-cells contribute to control of Mycobacterium tuberculosis infection, but little is known about the quality of the CD8 T-cell response in subjects with latent infection and in patients with active tuberculosis disease. CD8 T-cells recognizing epitopes from 6 different proteins of Mycobacterium tuberculosis were detected by tetramer staining. Intracellular cytokines staining for specific production of IFN-γ and IL-2 was performed, complemented by phenotyping of memory markers on antigen-specific CD8 T-cells. The ex-vivo frequencies of tetramer-specific CD8 T-cells in tuberculous patients before therapy were lower than in subjects with latent infection, but increased at four months after therapy to comparable percentages detected in subjects with latent infection. The majority of CD8 T-cells from subjects with latent infection expressed a terminally-differentiated phenotype (CD45RA⁺CCR7⁻). In contrast, tuberculous patients had only 35% of antigen-specific CD8 T-cells expressing this phenotype, while containing higher proportions of cells with an effector memory- and a central memory-like phenotype, and which did not change significantly after therapy. CD8 T-cells from subjects with latent infection showed a codominance of IL-2⁺/IFN-γ⁺ and IL-2⁻/IFN-γ⁺ T-cell populations; interestingly, only the IL-2⁺/IFN-γ⁺ population was reduced or absent in tuberculous patients, highly suggestive of a restricted functional profile of Mycobacterium tuberculosis-specific CD8 T-cells during active disease. These results suggest distinct Mycobacterium tuberculosis specific CD8 T-cell phenotypic and functional signatures between subjects which control infection (subjects with latent infection) and those who do not (patients with active disease).     

Patients with Tuberculosis Disease Have Mycobacterium tuberculosis-Specific CD8 T Cells with a Pro-Apoptotic Phenotype and Impaired Proliferative Capacity,Which Is Not Restored following Treatment

CD8 T cells play a critical role in control of chronic viral infections; however, the role of these cells in containing persistent bacterial infections, such as those caused by Mycobacterium tuberculosis (Mtb), is less clear. We assessed the phenotype and functional capacity of CD8 T cells specific for the immunodominant Mtb antigens CFP-10 and ESAT-6, in patients with pulmonary tuberculosis (TB) disease, before and after treatment, and in healthy persons with latent Mtb infection (LTBI). In patients with TB disease, CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells had an activated, pro-apoptotic phenotype, with lower Bcl-2 and CD127 expression, and higher Ki67, CD57, and CD95 expression, than in LTBI. When CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells were detectable, expression of distinct combinations of these markers was highly sensitive and specific for differentiating TB disease from LTBI. Successful treatment of disease resulted in changes of these markers, but not in restoration of CFP-10/ESAT-6-specific CD8 or CD4 memory T cell proliferative capacity. These data suggest that high mycobacterial load in active TB disease is associated with activated, short-lived CFP-10/ESAT-6-specific CD8 T cells with impaired functional capacity that is not restored following treatment. By contrast, LTBI is associated with preservation of long-lived CFP-10/ESAT-6-specific memory CD8 T cells that maintain high Bcl-2 expression and which may readily proliferate.     

Re-Evaluation of PD-1 Expression by T Cells as a Marker for Immune Exhaustion during SIV Infection

PD-1 expression is generally associated with exhaustion of T cells during chronic viral infections based on the finding that PD-1 expressing cells respond poorly to antigen activation and blockade of PD-1/PD-ligand interaction restores such antigen specific responses in vitro. We tested this hypothesis by examining PD-1 expression on virus-specific CD8 T cells and total T cells in vivo to determine whether PD-1 expression constitutes a reliable marker of immune exhaustion during SIV infection. The expression of PD-1 and Ki67 was monitored longitudinally on T cell subsets in peripheral blood, bone marrow, lymph node and rectal biopsy specimens from rhesus macaques prior to and post infection with pathogenic SIVmac239. During the course of infection, a progressive negative correlation was noted between PD-1 density and Ki67 expression in p11CM⁺ CD8⁺ T cells, as seen in other studies. However, for total and memory CD4 and CD8 T cells, a positive correlation was observed between PD-1 and Ki67 expression. Thus, while the levels of non-proliferating PD-1⁺ p11CM⁺ CD8 T cells were markedly elevated with progressing infection, such an increase was not seen on total T cells. In addition, total memory PD1⁺ T cells exhibited higher levels of CCR5 than PD-1⁻ T cells. Interestingly, few PD-1⁺ CD8⁺ T cells expressed CCR7 compared to PD-1⁺ CD4 T cells and PD-1⁻ T cells. In conclusion, overall PD1⁺ T cells likely represent a particular differentiation stage or trafficking ability rather than exhaustion and in the context of chronic SIV infection, the level of PD-1 expression by T cells does not by itself serve as a reliable marker for immune exhaustion.     

Lack of Evidence for mtDNA as a Biomarker of Innate Immune Activation in HIV Infection

Many human immunodeficiency virus (HIV) infected individuals suffer from persistent immune activation. Chronic inflammation and immune dysregulation have been associated with an increased risk of age-related diseases even among patients on highly active antiretroviral therapy. The factors leading to immune activation are complex, but have been hypothesized to include persistent viral replication with cellular death as well as microbial translocation across the gastrointestinal tract. Both processes may trigger innate immune responses since many native molecules released from dying cells are similar in structure to pathogen associated molecular patterns. These damage associated molecular patterns include mitochondrial DNA and formylated peptides. We hypothesized that circulating mitochondrial nucleic acid could serve as a biomarker for HIV-associated cell death and drive innate immune activation in infected individuals. We developed a quantitative polymerase chain reaction assay for plasma mitochondrial DNA and validated it on normal blood donors. We then measured mitochondrial DNA levels in acute and chronic HIV infection. While the assay proved to be accurate with a robust dynamic range, we did not find a significant association between HIV disease status and circulating mitochondrial DNA. We did, however, observe a negative correlation between age and plasma mitochondrial DNA levels in individuals with well-controlled HIV.     

Evidence for Generation of Suppressor T Cells in Mycobacterium lepraemurium-Infected CBA/J Mice,a Mouse Strain Highly Susceptible to the Infection

Susceptibility to Mycobacterium lepraemurium (MLM) infection markedly differed between two mouse strains, CBA/J and C57BL/6. CBA/J mice showed high susceptibility to MLM infection and developed either very weak or no delayed-type hypersensitivity (DTH) to MLM antigen after the injection of MLM. In contrast, C57BL/6 mice, which were resistant to MLM infection, showed significant DTH reaction to MLM antigen after the injection. Treatment of CBA/J mice with cyclophosphamide (Cy) conferred significant resistance to MLM infection on the CBA/J mice, and the treated mice developed a strong anti-MLM DTH response after the MLM injection. When spleen cells from MLM-infected CBA/J mice were transferred to Cy-treated and MLM-infected syngeneic mice, the anti-MLM DTH reaction of the recipient mice was suppressed. Treatment of the spleen cells to be transferred with anti-Thy-1.2 antibody or anti-I-J^k antiserum plus complement abrogated the suppressive activity. Thus, it is suggested that the high susceptibility of CBA/J mice to MLM infection is due to the generation of Cy-sensitive, I-J^k-positive suppressor T cells after infection with MLM.     

A Subset of Circulating Blood Mycobacteria-Specific CD4 T Cells Can Predict the Time to Mycobacterium tuberculosis Sputum Culture Conversion

We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR⁺Ki67⁺ on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67⁺HLA-DR⁺ Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67⁺HLA-DR⁺ T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ⁺IL2⁺TNFα⁺ CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67⁺HLA-DR⁺) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment.     

Characterization of a Cross-Reactive,Immunodominant and HLA-Promiscuous Epitope of Mycobacterium tuberculosis-Specific Major Antigenic Protein PPE68

PPE68 (Rv3873), a major antignic protein encoded by Mycobacteriun tuberculosis-specific genomic region of difference (RD)1, is a strong stimulator of peripheral blood mononuclear cells (PBMCs) obtained from tuberculosis patients and Mycobacterium bovis bacillus Calmette Guerin (BCG)-vaccianted healthy subjects in T helper (Th)1 cell assays, i.e. antigen-induced proliferation and interferon-gamma (IFN-γ) secretion. To confirm the antigen-specific recognition of PPE68 by T cells in IFN-γ assays, antigen-induced human T-cell lines were established from PBMCs of M. Bovis BCG-vaccinated and HLA-heterogeneous healthy subjects and tested with peptide pools of RD1 proteins. The results showed that PPE68 was recognized by antigen-specific T-cell lines from HLA-heteregeneous subjects. To further identify the immunodominant and HLA-promiscuous Th1-1 cell epitopes present in PPE68, 24 synthetic peptides covering the sequence of PPE68 were indivdually analyzed for HLA-DR binding prediction analysis and tested with PBMCs from M. bovis BCG-vaccinated and HLA-heterogeuous healthy subjects in IFN-γ assays. The results identified the peptide P9, i.e. aa 121-VLTATNFFGINTIPIALTEMDYFIR-145, as an immunodominant and HLA-DR promiscuous peptide of PPE68. Furthermore, by using deletion peptides, the immunodominant and HLA-DR promiscuous core sequence was mapped to aa 127-FFGINTIPIA-136. Interestingly, the core sequence is present in several PPE proteins of M. tuberculosis, and conserved in all sequenced strains/species of M. tuberculosis and M. tuberculosis complex, and several other pathogenic mycobacterial species, including M. leprae and M. avium-intracellulalae complex. These results suggest that the peptide aa 121–145 may be exploited as a peptide-based vaccine candidate against tuberculosis and other mycobacterial diseases.     

Therapeutic Memory T Cells Require Costimulation for Effective Clearance of a Persistent Viral Infection

Persistent viral infections are a major health concern worldwide. During persistent infection, overwhelming viral replication and the rapid loss of antiviral T-cell function can prevent immune-mediated clearance of the infection, and therapies to reanimate the immune response and purge persistent viruses have been largely unsuccessful. Adoptive immunotherapy using memory T cells is a highly successful therapeutic approach to eradicate a persistent viral infection. Understanding precisely how therapeutically administered memory T cells achieve clearance should improve our ability to terminate states of viral persistence in humans. Mice persistently infected from birth with lymphocytic choriomeningitis virus are tolerant to the pathogen at the T-cell level and thus provide an excellent model to evaluate immunotherapeutic regimens. Previously, we demonstrated that adoptively transferred memory T cells require recipient dendritic cells to effectively purge an established persistent viral infection. However, the mechanisms that reactivate and sustain memory T-cell responses during clearance of such an infection remain unclear. Here we establish that therapeutic memory T cells require CD80 and CD86 costimulatory signals to efficiently clear an established persistent viral infection in vivo. Early blockade of costimulatory pathways with CTLA-4-Fc decreased the secondary expansion of virus-specific CD8⁺ and CD4⁺ memory T cells as well as their ability to produce antiviral cytokines and purge the persistent infection. Late costimulation blockade also reduced virus-specific T-cell numbers, illustrating that sustained interactions with costimulatory molecules is required for efficient T-cell expansion. These findings indicate that antiviral memory T cells require costimulation to efficiently clear a persistent viral infection and that costimulatory pathways can be targeted to modulate the magnitude of an adoptive immunotherapeutic regimen.Persistent viruses, such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, cause major health problems worldwide and are extraordinarily difficult to clear following the establishment of persistence. Given the challenges associated with clearing persistent infections, it is important to develop and mechanistically understand therapeutic strategies that successfully achieve viral eradication without inducing permanent damage in the host. Studies using the lymphocytic choriomeningitis virus (LCMV) model system have convincingly demonstrated that a systemic persistent viral infection can be completely purged from a murine host by using a therapeutic approach referred to as adoptive immunotherapy (1, 15, 22, 29, 30). Remarkably, total body control of multiple persistent viral infections in both the mouse (1, 15, 22, 29, 30) and humans (8, 14, 24, 26, 31) can be achieved using adoptive immunotherapy. When mice are persistently infected at birth or in utero with LCMV (referred to as carrier mice), the virus establishes systemic persistence (6). Adult LCMV carrier mice are tolerant to the virus at the T-cell level and thus are unable to eradicate the pathogen (23), which provides an excellent model to study immunotherapeutic regimens. Immunocytotherapy relies on the adoptive transfer of virus-specific memory CD8 and CD4 T cells from LCMV-immune donor mice into recipient carrier mice (1, 15, 22, 29, 30). Following the therapeutic administration of memory cells, LCMV is purged from most peripheral tissues of carrier mice in 14 days, whereas more than 100 days are required to clear virus from the central nervous system (CNS) and kidneys (1, 15, 22). Furthermore, successful viral clearance requires antiviral “memory” but not “effector” T cells (11). Thus, in addition to its proven therapeutic relevance, this model also provides a paradigm to understand factors that regulate memory T cells following secondary exposure to pathogens in vivo.The mechanisms leading to activation of naïve T cells have been well described and involve recognition of major histocompatibility complex (MHC) peptide through the T-cell receptor (TCR) as well as costimulation (e.g., CD80 and CD86 interactions) (4, 25, 27). On the other hand, the factors that govern the activation and secondary expansion of memory CD8⁺ and CD4⁺ T cells are less clearly defined, particularly in an in vivo therapeutic setting. When memory T cells reencounter cognate antigen, they respond rapidly by producing cytokines and dividing. Previous studies indicated that there was no role for dendritic cells or costimulation (4, 27) in the reactivation of memory T cells; however, three recent studies have shown that dendritic cells (DCs) stimulate memory T-cell activity upon antigen rechallenge (2, 33) and during adoptive immunotherapy (15). Because MHC class I antigen (MHC-I) is expressed on nearly all cell types but costimulatory molecules are not, these three studies strongly suggested that DCs were influencing memory T cells with costimulatory pathways thought only to be required during priming. Indeed, when the issue was reexamined, it was revealed that memory CD8⁺ and CD4⁺ T cells require CD28-CD80/CD86 costimulation to be fully reactivated upon secondary exposure to antigen (3, 7, 21).Because therapeutically administered memory T cells require effective interactions with the host hematopoietic system (10), in particular dendritic cells (15), to achieve successful viral clearance, we set out to address several unanswered questions. First, is costimulation required for the immunotherapeutic clearance of an established persistent viral infection? This is a particularly important question because the requirements imposed on therapeutically administered memory T cells, which encounter immediate and overwhelmingly high levels of virus, heightened antigenic stimulation, and a unique inflammatory milieu, are likely to be different than those faced by endogenous memory T cells following pathogen rechallenge in an otherwise-quiescent environment. The second question we set out to address in this study was whether costimulation blockade could modulate the activities of an immunotherapeutic regimen consisting of memory T cells. This question is of great importance in a clinical setting where pathogen-specific memory T cells can induce severe tissue pathology through the release of effector molecules (12). Thus, it is critical to have a strategy to limit the magnitude of an undesirable response without impeding viral clearance.     

Viscoelasticity as a Biomarker for High-Throughput Flow Cytometry

The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods.     

Viscoelasticity as a Biomarker for High-Throughput Flow Cytometry

The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods.     

Clonal Expansions of CD8+ T Cells with IL-10 Secreting Capacity Occur during Chronic Mycobacterium tuberculosis Infection

The exact role of CD8⁺ T cells during Mycobacterium tuberculosis (Mtb) infection has been heavily debated, yet it is generally accepted that CD8⁺ T cells contribute to protection against Mtb. In this study, however, we show that the Mtb-susceptible CBA/J mouse strain accumulates large numbers of CD8⁺ T cells in the lung as infection progresses, and that these cells display a dysfunctional and immunosuppressive phenotype (PD-1⁺, Tim-3⁺, CD122⁺). CD8⁺ T cell expansions from the lungs of Mtb-infected CBA/J mice were also capable of secreting the immunosuppressive cytokine interleukin-10 (IL-10), although in vivo CD8⁺ T cell depletion did not significantly alter Mtb burden. Further analysis revealed that pulmonary CD8⁺ T cells from Mtb-infected CBA/J mice were clonally expanded, preferentially expressing T cell receptor (TcR) Vβ chain 8 (8.2, 8.3) or Vβ 14. Although Vβ8⁺ CD8⁺ T cells were responsible for the majority of IL-10 production, in vivo depletion of Vβ8⁺ did not significantly change the outcome of Mtb infection, which we hypothesize was a consequence of their dual IL-10/IFN-γ secreting profiles. Our data demonstrate that IL-10-secreting CD8⁺ T cells can arise during chronic Mtb infection, although the significance of this T cell population in tuberculosis pathogenesis remains unclear.     

In Silico Model as a Tool for Interpretation of Intestinal Infection Studies

In nutrition research the number of human in vivo experiments is limited because of the many restrictions and the high costs of testing in humans. Up to now predictive computer models aiming to enhance research have been rare or too complex, with many nonmeasurable adjustable parameters. This study aimed to develop a basic physicochemical computer model for a first quantitative interpretation of results obtained from in vivo intestinal experiments with bacteria. This new modeling approach is validated with results obtained from gut infection studies in vivo. The design of the model is described, and its ability to reproduce experimental data is evaluated. The model predictions are compared with new experimental data. The phenomena that take place in the gastrointestinal tract are summarized by model constants for growth, adherence, and release of bacteria. Although the model is far from describing all details and many processes in the intestine are combined, the model calculation results lead to reasonable conclusions and interesting hypotheses. One of these hypotheses concluded from the model outcomes is that Escherichia coli bacteria have a much lower intestinal growth rate in humans than in rats. Extra laboratory validation experiments proved the reliability of this hypothesis predicted by the model. In addition, the known protective effect of dietary calcium and detrimental effect of clindamycin on the growth and adherence of Salmonella bacteria could be quantified. From these results it is clear that the model enhances the interpretation of in vivo gastrointestinal experiments and will facilitate research trajectories towards new functional foods that improve resistance to pathogenic bacteria in humans.     

TIGIT Marks Exhausted T Cells,Correlates with Disease Progression,and Serves as a Target for Immune Restoration in HIV and SIV Infection

HIV infection induces phenotypic and functional changes to CD8⁺ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8⁺ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT⁺ and TIGIT⁺ PD-1⁺ CD8⁺ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8⁺ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8⁺ T cell effector responses. The frequency of TIGIT⁺ CD4⁺ T cells correlated with the CD4⁺ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.     

DNA Methylation as a Biomarker for Cardiovascular Disease Risk

Background

Elevated serum homocysteine is associated with an increased risk of cardiovascular disease (CVD). This may reflect a reduced systemic remethylation capacity, which would be expected to cause decreased genomic DNA methylation in peripheral blood leukocytes (PBL).

Methodology/Principal Findings

We examined the association between prevalence of CVD (myocardial infarction, stroke) and its predisposing conditions (hypertension, diabetes) and PBL global genomic DNA methylation as represented by ALU and Satellite 2 (AS) repetitive element DNA methylation in 286 participants of the Singapore Chinese Health Study, a population-based prospective investigation of 63,257 men and women aged 45–74 years recruited during 1993–1998. Men exhibited significantly higher global DNA methylation [geometric mean (95% confidence interval (CI)): 159 (143, 178)] than women [133 (121, 147)] (P = 0·01). Global DNA methylation was significantly elevated in men with a history of CVD or its predisposing conditions at baseline (P = 0·03) but not in women (P = 0·53). Fifty-two subjects (22 men, 30 women) who were negative for these CVD/predisposing conditions at baseline acquired one or more of these conditions by the time of their follow-up I interviews, which took place on average about 5·8 years post-enrollment. Global DNA methylation levels of the 22 incident cases in men were intermediate (AS, 177) relative to the 56 male subjects who remained free of CVD/predisposing conditions at follow-up (lowest AS, 132) and the 51 male subjects with a diagnosis of CVD or predisposing conditions reported at baseline (highest AS 184) (P for trend = 0.0008) No such association was observed in women (P = 0.91). Baseline body mass index was positively associated with AS in both men and women (P = 0·007).

Conclusions/Significance

Our findings indicate that elevated, not decreased, PBL DNA methylation is positively associated with prevalence of CVD/predisposing conditions and obesity in Singapore Chinese.     

Effortless Attention as a Biomarker for Experienced Mindfulness Practitioners

Objective

The present study aimed at comparing frontal beta power between long-term (LTM) and first-time meditators (FTM), before, during and after a meditation session. We hypothesized that LTM would present lower beta power than FTM due to lower effort of attention and awareness.

Methods

Twenty one participants were recruited, eleven of whom were long-term meditators. The subjects were asked to rest for 4 minutes before and after open monitoring (OM) meditation (40 minutes).

Results

The two-way ANOVA revealed an interaction between the group and moment factors for the Fp1 (p<0.01), F7 (p = 0.01), F3 (p<0.01), Fz (p<0.01), F4 (p<0.01), F8 (p<0.01) electrodes.

Conclusion

We found low power frontal beta activity for LTM during the task and this may be associated with the fact that OM is related to bottom-up pathways that are not present in FTM.

Significance

We hypothesized that the frontal beta power pattern may be a biomarker for LTM. It may also be related to improving an attentive state and to the efficiency of cognitive functions, as well as to the long-term experience with meditation (i.e., life-time experience and frequency of practice).