Phenotypic and Functional Properties of Helios+ Regulatory T Cells

Helios, an Ikaros family transcription factor, is preferentially expressed at the mRNA and protein level in regulatory T cells. Helios expression previously appeared to be restricted to thymic-derived Treg. Consistent with recent data, we show here that Helios expression is inducible in vitro under certain conditions. To understand phenotypic and functional differences between Helios+ and Helios− Treg, we profiled cell-surface markers of FoxP3+ Treg using unmanipulated splenocytes. We found that CD103 and GITR are expressed at high levels on a subset of Helios+ Treg and that a Helios+ Treg population could be significantly enriched by FACS sorting using these two markers. Quantitative real-time PCR (qPCR) analysis revealed increased TGF-β message in Helios+ Treg, consistent with the possibility that this population possesses enhanced regulatory potential. In tumor-bearing mice, we found that Helios+ Treg were relatively over-represented in the tumor-mass, and BrdU studies showed that, in vivo, Helios+ Treg proliferated more than Helios− Treg. We hypothesized that Helios-enriched Treg might exert increased suppressive effects. Using in vitro suppression assays, we show that Treg function correlates with the absolute number of Helios+ cells in culture. Taken together, these data show that Helios+ Treg represent a functional subset with associated CD103 and GITR expression

Treatment outcome and associated factors of severe acute malnutrition among 6–59 months old children in Debre Markos and Finote Selam hospitals, Northwest Ethiopia: a retrospective cohort study

BACKGROUND: In Ethiopia, the health sector has increased its efforts to enhance good nutritional practices through health education, treatment of extremely malnourished children and provision of micronutrients for mothers and children. But, the poor nutritional status of women and children continues to be still a major public health problem. METHODS: A retrospective cohort study was conducted to assess the treatment outcome and associated factors of severe acute malnutrition among a total of 253 children age 6–59 months old. Severe acute malnutrition registration logbook and patient charts were used as a source of data. Data were entered in to Epi-data version 3.1 and exported to SPSS version 20 for analysis. To identify associated factors, Cox proportional hazard analysis was computed and p-value <0.05 at 95% confidence interval was considered as statistically significant. RESULTS: The recovery rate was 77.9% and the overall median recovery time was 11 days. Those children age from 24 to 35 months had 34% lower probability of recovery from SAM compared to 6–11 months old children (AHR = 0.66, 95% CI: 0.35–0.89). Children whose ages from 36 to 59 months had 47% lower probability of recovery from SAM compared to 6–11 months old children (AHR = 0.53, 95% CI: 0.31–0.91). HIV negative children had 2.48 times higher probability of getting recovered from SAM compared to HIV positive children (AHR = 2.48, 95% CI: 1.23–5.01). Children who didn’t take folic acid supplement had 65% lower probability of recovery from SAM compared to children who took folic acid supplement (AHR = 0.35, 95% CI: 0.14–0.89). CONCLUSIONS: This study found that recovery rate of 6–59 months old children treated for severe acute malnutrition in therapeutics units was in acceptable range based on the WHO recommendation. Folic acid supplementation and screening for HIV status should be promoted at all levels of health facilities during early age

Epigenetic biotypes of post-traumatic stress disorder in war-zone exposed veteran and active duty males

Post-traumatic stress disorder (PTSD) is a heterogeneous condition evidenced by the absence of objective physiological measurements applicable to all who meet the criteria for the disorder as well as divergent responses to treatments. This study capitalized on biological diversity observed within the PTSD group observed following epigenome-wide analysis of a well-characterized Discovery cohort (N = 166) consisting of 83 male combat exposed veterans with PTSD, and 83 combat veterans without PTSD in order to identify patterns that might distinguish subtypes. Computational analysis of DNA methylation (DNAm) profiles identified two PTSD biotypes within the PTSD+ group, G1 and G2, associated with 34 clinical features that are associated with PTSD and PTSD comorbidities. The G2 biotype was associated with an increased PTSD risk and had higher polygenic risk scores and a greater methylation compared to the G1 biotype and healthy controls. The findings were validated at a 3-year follow-up (N = 59) of the same individuals as well as in two independent, veteran cohorts (N = 54 and N = 38), and an active duty cohort (N = 133). In some cases, for example Dopamine-PKA-CREB and GABA-PKC-CREB signaling pathways, the biotypes were oppositely dysregulated, suggesting that the biotypes were not simply a function of a dimensional relationship with symptom severity, but may represent distinct biological risk profiles underpinning PTSD. The identification of two novel distinct epigenetic biotypes for PTSD may have future utility in understanding biological and clinical heterogeneity in PTSD and potential applications in risk assessment for active duty military personnel under non-clinician-administered settings, and improvement of PTSD diagnostic markers

The role of PD-1 in human CD8 prostate infiltrating lymphocytes. (66.12)

Abstract CD8 T cell tolerance to tumors is multi faceted and includes both extrinsic and intrinsic factors. Extrinsically, the tumor microenvironment seems to play a large role in the functional inertness of CD8 T cells; major contributors are persistent antigen exposure in the microenvironment as well as T regulatory cells. Intrinsically, lack of costimulation seems to play a role (anergy), and often, the presence of inhibitory checkpoints such as PD-1, CTLA-4, LAG-3, and 41BB can also facilitate the down regulation of CD8 T cell function. In CD8 prostate infiltrating lymphocytes (PILS) from cancer patients, we observe refractoriness to TCR stimulation accompanied by reduced cytokine production (IFN-γ and TNF-α) that cannot be reversed by addition of IL-2. Thus, in trying to decipher the exact intrinsic factor that could play a direct role on the CD8 PILS, we measured inhibitory checkpoints on the cell surface. PD-1 was a major checkpoint, and seemed to correlate with reduced Granzyme B production. Interestingly, the total CD8 PIL population was not deficient in Granzyme B production, just the PD-1+ CD8 PILS. Additionally, blockade with anti PD-1 restored proliferation and Granzyme B production in the CD8 PILS, which indicates a potential clinical implication of anti PD-1 immunotherapy.</jats:p

A multi-omic analysis of human naïve CD4+ T cells

Background: Cellular function and diversity are orchestrated by complex interactions of fundamental biomolecules including DNA, RNA and proteins. Technological advances in genomics, epigenomics, transcriptomics and proteomics have enabled massively parallel and unbiased measurements. Such high-throughput technologies have been extensively used to carry out broad, unbiased studies, particularly in the context of human diseases. Nevertheless, a unified analysis of the genome, epigenome, transcriptome and proteome of a single human cell type to obtain a coherent view of the complex interplay between various biomolecules has not yet been undertaken. Here, we report the first multi-omic analysis of human primary naïve CD4+ T cells isolated from a single individual. Results: Integrating multi-omics datasets allowed us to investigate genome-wide methylation and its effect on mRNA/protein expression patterns, extent of RNA editing under normal physiological conditions and allele specific expression in naïve CD4+ T cells. In addition, we carried out a multi-omic comparative analysis of naïve with primary resting memory CD4+ T cells to identify molecular changes underlying T cell differentiation. This analysis provided mechanistic insights into how several molecules involved in T cell receptor signaling are regulated at the DNA, RNA and protein levels. Phosphoproteomics revealed downstream signaling events that regulate these two cellular states. Availability of multi-omics data from an identical genetic background also allowed us to employ novel proteogenomics approaches to identify individual-specific variants and putative novel protein coding regions in the human genome. Conclusions: We utilized multiple high-throughput technologies to derive a comprehensive profile of two primary human cell types, naïve CD4+ T cells and memory CD4+ T cells, from a single donor. Through vertical as well as horizontal integration of whole genome sequencing, methylation arrays, RNA-Seq, miRNA-Seq, proteomics, and phosphoproteomics, we derived an integrated and comparative map of these two closely related immune cells and identified potential molecular effectors of immune cell differentiation following antigen encounter

Functional Heatmap: an automated and interactive pattern recognition tool to integrate time with multi-omics assays

Abstract Background Life science research is moving quickly towards large-scale experimental designs that are comprised of multiple tissues, time points, and samples. Omic time-series experiments offer answers to three big questions: what collective patterns do most analytes follow, which analytes follow an identical pattern or synchronize across multiple cohorts, and how do biological functions evolve over time. Existing tools fall short of robustly answering and visualizing all three questions in a unified interface. Results Functional Heatmap offers time-series data visualization through a Master Panel page, and Combined page to answer each of the three time-series questions. It dissects the complex multi-omics time-series readouts into patterned clusters with associated biological functions. It allows users to identify a cascade of functional changes over a time variable. Inversely, Functional Heatmap can compare a pattern with specific biology respond to multiple experimental conditions. All analyses are interactive, searchable, and exportable in a form of heatmap, line-chart, or text, and the results are easy to share, maintain, and reproduce on the web platform. Conclusions Functional Heatmap is an automated and interactive tool that enables pattern recognition in time-series multi-omics assays. It significantly reduces the manual labour of pattern discovery and comparison by transferring statistical models into visual clues. The new pattern recognition feature will help researchers identify hidden trends driven by functional changes using multi-tissues/conditions on a time-series fashion from omic assays

Altered Fecal Microbiota and Urine Metabolome as Signatures of Soman Poisoning

AbstractThe experimental pathophysiology of organophosphorus (OP) chemical exposure has been extensively reported. Here, we describe an altered fecal microbiota and urine metabolome that follows intoxication with soman, a lipophilic G class chemical warfare nerve agent. Non-anaesthetized Sprague-Dawley male rats were subcutaneously administered soman at 0.8 - 1.0 of the median lethal dose (LD50) and evaluated for signs of toxicity. Animals were stratified based on seizing activity to evaluate effects of soman exposure on fecal bacterial biota and urine metabolites. Soman exposure reshaped fecal bacterial biota by preferentially expandingFacklamia, Agrobacterium,Bilophila,Enterobacter, andMorganellagenera of theFirmicutesandProteobacteriaphyla, some of which are known to hydrolyze OPs. However, analogous changes were not observed in the bacterial biota of the ileum, which remained the same irrespective of dose or seizing status of animals after exposure. Interestingly, when considering just the seizing status of animals, we found that the urine metabolome was markedly altered. Leukotriene C4, kynurenic acid, 5-hydroxyindoleacetic acid, norepinephrine, and aldosterone were excreted at much higher rates at 72 hrs in seizing animals, consistent with early multi-organ involvement during soman poisoning. However, at 75 days post soman exposure, bacterial biota stabilized and no differences were observed. These findings demonstrate the feasibility of using the dysbiosis of fecal bacterial biota in combination with urine metabolome alterations as forensic evidence for OP exposure temporally.ImportanceThe paucity of assays to determine physiologically relevant OP exposure presents an opportunity to explore the use bacterial sentinels in combination with urine to assess changes in the exposed host. Recent advances in technologies and computational approaches have enabled researches to survey large community level changes of gut bacterial biota and metabolomic changes in various biospecimens. Here, we profile combined changes in bacterial biota and urine metabolome due to chemical warfare OP exposure. The significance of our work is to reveal that monitoring bacterial biota and urine metabolites as surrogates of OP exposure in biospecimens suitable for existing clinical laboratory workflows is plausible without the need for the development of new technology, invasive procedures, or complicated analytical approaches. The larger value of such an approach is that any setting with a moderate clinical chemistry and microbiology capability can determine pre-symptomatic exposure to enhance current triage standards in case of mass exposures, refugee movements, humanitarian missions, and training settings once an algorithm has been validated. In the event of “potential” exposures by time or distance, this assay can be further developed to estimate affected radius or time dimension for health monitoring and treatment interventions.</jats:sec

Role of human lymphocyte activation gene 3 in tumor infiltrating lymphocytes (101.12)

Abstract Among the various molecules that regulate T cell function, lymphocyte activation gene 3 (LAG- 3) has garnered significant interest. LAG-3 is expressed on activated T cells, B cells, NK cells, tumor infiltrating lymphocytes (TILs), and plasmacytoid dendritic cells. We previously showed that LAG-3 was relatively over-expressed on HA-specific transgenic T cells rendered anergic in vivo by encounter with cognate self antigen. In this system, regulatory activity could be functionally blocked with a LAG-3 specific monoclonal antibody (Huang et al). Observations in our lab using LAG-3 knockout mice demonstrate that CD8 T cells undergo enhanced homeostatic proliferation in vivo if LAG-3 is absent. Currently, we are conducting studies to understand the role of LAG-3 in human cancer. Via microarray, we compared CD4+25+GITR+ (Treg) T cells from the prostate to CD4+25-45RA+ (naïve) T cells from the peripheral blood. We found LAG-3 and CTLA-4 to be relatively upregulated in prostate infiltrating T regulatory cells. Also, at the expression level, we observed variable levels of LAG-3 expression on patient CD8 tumor infiltrating T lymphocytes (TILs). At a functional level, we have shown that a human anti-human LAG-3 antibody enhances T cell proliferation and cytokine function in a mixed lymphocyte reaction. Further studies that are currently underway suggest that LAG-3 could be a promising candidate for enhancing anti-tumor immunotherapy in a clinical setting.</jats:p

Anti-tumor effects of endogenous prostate cancer-specific CD8 T cells in a murine TCR transgenic model

BACKGROUND: The CD8 T cell response to prostate and other cancers is often functionally diminished or absent. This may occur via deletion of tumor-specific T cells, through acquisition of an anergic phenotype, or via active suppression mediated by another population of cells. METHODS: We used a double transgenic model in which mice express CD8 T cells specific for a prostate/prostate cancer antigen to study the response of CD8 T cells to evolving autochronous prostate tumors in TRAMP mice. CD8 T cells were analyzed for functionality by measuring IFN-γ production via flow cytometry and via an in vivo CTL killing assay. In addition, pathological scoring of the prostates of the double transgenic mice was compared to scoring of tumor burden prostates of ProTRAMP mice. RESULTS: Tumor-specific CD8 T cells were not grossly deleted in these animals, but evidenced a clearly non-functional phenotype. Interestingly, full lytic function was rapidly recovered upon removal from tumor-bearing mice. CONCLUSIONS: These data indicate a role for continuous antigen exposure in the maintenance of tumor-specific CD8 T cell tolerance to prostate cancer