Human TSCM cell dynamics in vivo are compatible with long-lived immunological memory and stemness.

Adaptive immunity relies on the generation and maintenance of memory T cells to provide protection against repeated antigen exposure. It has been hypothesised that a self-renewing population of T cells, named stem cell-like memory T (TSCM) cells, are responsible for maintaining memory. However, it is not clear if the dynamics of TSCM cells in vivo are compatible with this hypothesis. To address this issue, we investigated the dynamics of TSCM cells under physiological conditions in humans in vivo using a multidisciplinary approach that combines mathematical modelling, stable isotope labelling, telomere length analysis, and cross-sectional data from vaccine recipients. We show that, unexpectedly, the average longevity of a TSCM clone is very short (half-life < 1 year, degree of self-renewal = 430 days): far too short to constitute a stem cell population. However, we also find that the TSCM population is comprised of at least 2 kinetically distinct subpopulations that turn over at different rates. Whilst one subpopulation is rapidly replaced (half-life = 5 months) and explains the rapid average turnover of the bulk TSCM population, the half-life of the other TSCM subpopulation is approximately 9 years, consistent with the longevity of the recall response. We also show that this latter population exhibited a high degree of self-renewal, with a cell residing without dying or differentiating for 15% of our lifetime. Finally, although small, the population was not subject to excessive stochasticity. We conclude that the majority of TSCM cells are not stem cell-like but that there is a subpopulation of TSCM cells whose dynamics are compatible with their putative role in the maintenance of T cell memory.

The relative contributions of infectious and mitotic spread to HTLV-1 persistence.

Human T-lymphotropic virus type-1 (HTLV-1) persists within hosts via infectious spread (de novo infection) and mitotic spread (infected cell proliferation), creating a population structure of multiple clones (infected cell populations with identical genomic proviral integration sites). The relative contributions of infectious and mitotic spread to HTLV-1 persistence are unknown, and will determine the efficacy of different approaches to treatment. The prevailing view is that infectious spread is negligible in HTLV-1 persistence beyond early infection. However, in light of recent high-throughput data on the abundance of HTLV-1 clones, and recent estimates of HTLV-1 clonal diversity that are substantially higher than previously thought (typically between 104 and 105 HTLV-1+ T cell clones in the body of an asymptomatic carrier or patient with HTLV-1-associated myelopathy/tropical spastic paraparesis), ongoing infectious spread during chronic infection remains possible. We estimate the ratio of infectious to mitotic spread using a hybrid model of deterministic and stochastic processes, fitted to previously published HTLV-1 clonal diversity estimates. We investigate the robustness of our estimates using three alternative estimators. We find that, contrary to previous belief, infectious spread persists during chronic infection, even after HTLV-1 proviral load has reached its set point, and we estimate that between 100 and 200 new HTLV-1 clones are created and killed every day. We find broad agreement between all estimators. The risk of HTLV-1-associated malignancy and inflammatory disease is strongly correlated with proviral load, which in turn is correlated with the number of HTLV-1-infected clones, which are created by de novo infection. Our results therefore imply that suppression of de novo infection may reduce the risk of malignant transformation.

Different Selected Mechanisms Attenuated the Inhibitory Interaction of KIR2DL1 with C2+ HLA-C in Two Indigenous Human Populations in Southern Africa.

The functions of human NK cells in defense against pathogens and placental development during reproduction are modulated by interactions of killer cell Ig-like receptors (KIRs) with HLA-A, -B and -C class I ligands. Both receptors and ligands are highly polymorphic and exhibit extensive differences between human populations. Indigenous to southern Africa are the KhoeSan, the most ancient group of modern human populations, who have highest genomic diversity worldwide. We studied two KhoeSan populations, the Nama pastoralists and the ≠Khomani San hunter-gatherers. Comprehensive next-generation sequence analysis of HLA-A, -B, and -C and all KIR genes identified 248 different KIR and 137 HLA class I, which assort into ∼200 haplotypes for each gene family. All 74 Nama and 78 ≠Khomani San studied have different genotypes. Numerous novel KIR alleles were identified, including three arising by intergenic recombination. On average, KhoeSan individuals have seven to eight pairs of interacting KIR and HLA class I ligands, the highest diversity and divergence of polymorphic NK cell receptors and ligands observed to date. In this context of high genetic diversity, both the Nama and the ≠Khomani San have an unusually conserved, centromeric KIR haplotype that has arisen to high frequency and is different in the two KhoeSan populations. Distinguishing these haplotypes are independent mutations in KIR2DL1, which both prevent KIR2DL1 from functioning as an inhibitory receptor for C2+ HLA-C. The relatively high frequency of C2+ HLA-C in the Nama and the ≠Khomani San appears to have led to natural selection against strong inhibitory C2-specific KIR.

HIV-1 adaptation to NK cell-mediated immune pressure.

The observation, by Alter et al., of the enrichment of NK cell "escape" variants in individuals carrying certain Killer-cell Immunoglobulin-like Receptor (KIR) genes is compelling evidence that natural killer (NK) cells exert selection pressure on HIV-1. Alter et al hypothesise that variant peptide, in complex with HLA class I molecules binds KIR receptors and either increases NK cell inhibition or decreases NK cell activation compared to wild type peptide thus leading to virus escape from the NK cell response. According to this hypothesis, in order for NK cells to select for an escape variant, an individual must carry both the KIR and an HLA ligand that binds the variant peptide. In this study we estimate the proportion of the population that is capable of selecting for escape variants and use both epidemiological modelling and a model-free approach to investigate whether this proportion explains the observed variant enrichment. We found that the fraction of individuals within whom the variant would have a selective advantage was low and was unable to explain the high degree of enrichment observed. We conclude that whilst Alter et al's data is consistent with selection pressure, the mechanism that they postulate is unlikely. The importance of this work is two-fold. Firstly, it forces a re-evaluation of some of the clearest evidence that NK cells exert a protective effect in HIV-1 infection. Secondly, it implies that there is a significant aspect of immunology that is not understood: it is possible that KIRs bind much more widely than was previously appreciated; that a gene in linkage with the KIR genes is responsible for considerable peptide-dependent selection or that variant peptides are indirectly impacting KIR ligation.

PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells.

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

BIITE: A Tool to Determine HLA Class II Epitopes from T Cell ELISpot Data.

Activation of CD4+ T cells requires the recognition of peptides that are presented by HLA class II molecules and can be assessed experimentally using the ELISpot assay. However, even given an individual's HLA class II genotype, identifying which class II molecule is responsible for a positive ELISpot response to a given peptide is not trivial. The two main difficulties are the number of HLA class II molecules that can potentially be formed in a single individual (3-14) and the lack of clear peptide binding motifs for class II molecules. Here, we present a Bayesian framework to interpret ELISpot data (BIITE: Bayesian Immunogenicity Inference Tool for ELISpot); specifically BIITE identifies which HLA-II:peptide combination(s) are immunogenic based on cohort ELISpot data. We apply BIITE to two ELISpot datasets and explore the expected performance using simulations. We show this method can reach high accuracies, depending on the cohort size and the success rate of the ELISpot assay within the cohort.

Single nucleotide polymorphism array profiling identifies distinct chromosomal aberration patterns across colorectal adenomas and carcinomas.

The progression of benign colorectal adenomas into cancer is associated with the accumulation of chromosomal aberrations. Even though patterns and frequencies of chromosomal aberrations have been well established in colorectal carcinomas, corresponding patterns of aberrations in adenomas are less well documented. The aim of this study was to profile chromosomal aberrations across colorectal adenomas and carcinomas to provide a better insight into key changes during tumor initiation and progression. Single nucleotide polymorphism array analysis was performed on 216 colorectal tumor/normal matched pairs, comprising 60 adenomas and 156 carcinomas. While many chromosomal aberrations were specific to carcinomas, those with the highest frequency in carcinomas (amplification of chromosome 7, 13q, and 20q; deletion of 17p and chromosome 18; LOH of 1p, chromosome 4, 5q, 8p, 17p, chromosome 18, and 20p) were also identified in adenomas. Hierarchical clustering using chromosomal aberrations revealed three distinct subtypes. Interestingly, these subtypes were only partially dependent on tumor staging. A cluster of colorectal cancer patients with frequent chromosomal deletions had the least favorable prognosis, and a number of adenomas (n = 9) were also present in the cluster suggesting that, at least in some tumors, the chromosomal aberration pattern is determined at a very early stage of tumor formation. Finally, analysis of LOH events revealed that copy-neutral/gain LOH (CN/G-LOH) is frequent (>10%) in carcinomas at 5q, 11q, 15q, 17p, chromosome 18, 20p, and 22q. Deletion of the corresponding region is sometimes present in adenomas, suggesting that LOH at these loci may play an important role in tumor initiation.

Transmission of Hepatitis C Virus among Prisoners, Australia, 2005-2012.

Hepatitis C virus (HCV) is predominantly transmitted between persons who inject drugs. For this population, global prevalence of HCV infection is high and incarceration is common and an independent risk factor for HCV acquisition. To explore HCV transmission dynamics in incarcerated populations, we integrated virus sequences with risk behavior and spatiotemporal data and analyzed transmission clusters among prisoners in Australia. We detected 3 clusters of recent HCV transmission consisting of 4 likely in-custody transmission events involving source/recipient pairs located in the same prison at the same time. Of these 4 events, 3 were associated with drug injecting and equipment sharing. Despite a large population of prisoners with chronic HCV, recent transmission events were identified in the prison setting. This ongoing HCV transmission among high-risk prisoners argues for expansion of prevention programs to reduce HCV transmission in prisons.

Activity of a heptad of transcription factors is associated with stem cell programs and clinical outcome in acute myeloid leukemia.

Aberrant transcriptional programs in combination with abnormal proliferative signaling drive leukemic transformation. These programs operate in normal hematopoiesis where they are involved in hematopoietic stem cell (HSC) proliferation and maintenance. Ets Related Gene (ERG) is a component of normal and leukemic stem cell signatures and high ERG expression is a risk factor for poor prognosis in acute myeloid leukemia (AML). However, mechanisms that underlie ERG expression in AML and how its expression relates to leukemic stemness are unknown. We report that ERG expression in AML is associated with activity of the ERG promoters and +85 stem cell enhancer and a heptad of transcription factors that combinatorially regulate genes in HSCs. Gene expression signatures derived from ERG promoter-stem cell enhancer and heptad activity are associated with clinical outcome when ERG expression alone fails. We also show that the heptad signature is associated with AMLs that lack somatic mutations in NPM1 and confers an adverse prognosis when associated with FLT3 mutations. Taken together, these results suggest that transcriptional regulators cooperate to establish or maintain primitive stem cell-like signatures in leukemic cells and that the underlying pattern of somatic mutations contributes to the development of these signatures and modulate their influence on clinical outcome.

Quantitative analysis of three-dimensional fluorescence localization microscopy data.

Identifying the three-dimensional molecular organization of subcellular organelles in intact cells has been challenging to date. Here we present an analysis approach for three-dimensional localization microscopy that can not only identify subcellular objects below the diffraction limit but also quantify their shape and volume. This approach is particularly useful to map the topography of the plasma membrane and measure protein distribution within an undulating membrane.

Signal analysis for genome-wide maps of histone modifications measured by ChIP-seq.

MOTIVATION: Chromatin structure, including post-translational modifications of histones, regulates gene expression, alternative splicing and cell identity. ChIP-seq is an increasingly used assay to study chromatin function. However, tools for downstream bioinformatics analysis are limited and are only based on the evaluation of signal intensities. We reasoned that new methods taking into account other signal characteristics such as peak shape, location and frequencies might reveal new insights into chromatin function, particularly in situation where differences in read intensities are subtle. RESULTS: We introduced an analysis pipeline, based on linear predictive coding (LPC), which allows the capture and comparison of ChIP-seq histone profiles. First, we show that the modeled signal profiles distinguish differentially expressed genes with comparable accuracy to signal intensities. The method was robust against parameter variations and performed well up to a signal-to-noise ratio of 0.55. Additionally, we show that LPC profiles of activating and repressive histone marks cluster into distinct groups and can be used to predict their function. AVAILABILITY AND IMPLEMENTATION: http://www.cancerresearch.unsw.edu.au/crcweb.nsf/page/LPCHP A Matlab implementation along with usage instructions and an example input file are available from: http://www.cancerresearch.unsw.edu.au/crcweb.nsf/page/LPCHP.

KIR-HLA interactions extend human CD8+ T cell lifespan in vivo.

BACKGROUNDThere is increasing evidence, in transgenic mice and in vitro, that inhibitory killer cell immunoglobulin-like receptors (iKIRs) can modulate T cell responses. Furthermore, we have previously shown that iKIRs are an important determinant of T cell-mediated control of chronic viral infection and that these results are consistent with an increase in the CD8+ T cell lifespan due to iKIR-ligand interactions. Here, we tested this prediction and investigated whether iKIRs affect T cell lifespan in humans in vivo.METHODSWe used stable isotope labeling with deuterated water to quantify memory CD8+ T cell survival in healthy individuals and patients with chronic viral infections.RESULTSWe showed that an individual's iKIR-ligand genotype was a significant determinant of CD8+ T cell lifespan: in individuals with 2 iKIR-ligand gene pairs, memory CD8+ T cells survived, on average, for 125 days; in individuals with 4 iKIR-ligand gene pairs, the memory CD8+ T cell lifespan doubled to 250 days. Additionally, we showed that this survival advantage was independent of iKIR expression by the T cell of interest and, further, that the iKIR-ligand genotype altered the CD8+ and CD4+ T cell immune aging phenotype.CONCLUSIONSTogether, these data reveal an unexpectedly large effect of iKIR genotype on T cell survival.FUNDINGWellcome Trust; Medical Research Council; EU Horizon 2020; EU FP7; Leukemia and Lymphoma Research; National Institute of Health Research (NIHR) Imperial Biomedical Research Centre; Imperial College Research Fellowship; National Institutes of Health; Jefferiss Trust.

Identification of proliferative and non-proliferative subpopulations of leukemic cells in CLL.

Pathogenesis in chronic lymphocytic leukemia (CLL) is strongly linked to the potential for leukemic cells to migrate to and proliferate within lymph-nodes. Previous in vivo studies suggest that all leukemic cells participate in cycles of migration and proliferation. In vitro studies, however, have shown heterogeneous migration patterns.To investigate tumor subpopulation kinetics, we performed in vivo isotope-labeling studies in ten patients with IgVH-mutated CLL (M-CLL). Using deuterium-labeled glucose, we investigated proliferation in sub-populations defined by CXCR4/CD5 and surface (sIgM) expression. Mathematical modeling was performed to test the likelihood that leukemic cells exist as distinct sub-populations or as a single population with the same proliferative capacity. Further labeling studies in two patients with M-CLL commencing idelalisib investigated the effect of B-cell receptor (BCR) antagonists on sub-population kinetics.Modeling revealed that data were more consistent with a model comprising distinct sub-populations (p = 0.008) with contrasting, characteristic kinetics. Following idelalisib therapy, similar labeling suppression across all sub-populations suggested that the most proliferative subset is the most sensitive to treatment. As the quiescent sub-population precedes treatment, selection likely explains the persistence of such residual non-proliferating populations during BCR-antagonist therapy. These findings have clinical implications for discontinuation of long-term BCR-antagonist treatment in selected patients.

Identifying the immune interactions underlying HLA class I disease associations.

Variation in the risk and severity of many autoimmune diseases, malignancies and infections is strongly associated with polymorphisms at the HLA class I loci. These genetic associations provide a powerful opportunity for understanding the etiology of human disease. HLA class I associations are often interpreted in the light of 'protective' or 'detrimental' CD8+ T cell responses which are restricted by the host HLA class I allotype. However, given the diverse receptors which are bound by HLA class I molecules, alternative interpretations are possible. As well as binding T cell receptors on CD8+ T cells, HLA class I molecules are important ligands for inhibitory and activating killer immunoglobulin-like receptors (KIRs) which are found on natural killer cells and some T cells; for the CD94:NKG2 family of receptors also expressed mainly by NK cells and for leukocyte immunoglobulin-like receptors (LILRs) on myeloid cells. The aim of this study is to develop an immunogenetic approach for identifying and quantifying the relative contribution of different receptor-ligand interactions to a given HLA class I disease association and then to use this approach to investigate the immune interactions underlying HLA class I disease associations in three viral infections: Human T cell Leukemia Virus type 1, Human Immunodeficiency Virus type 1 and Hepatitis C Virus as well as in the inflammatory condition Crohn's disease.

When considering someone's risk of disease, every person is different but some similarities can be found when looking across populations. Some people are more likely to develop a certain disease, while others are protected in some way. Part of this variation is explained by the individual's genes, while their lifestyle and environment are other factors. Numerous studies have looked for associations between different versions of genes, known as gene variants, and the occurrence of disease to identify who is at risk. There is one cluster of genes called the HLA genes that is a well-known hotspot for disease associations. The HLA cluster is named for the group of proteins it encodes, called the human leukocyte antigen (HLA) complex. These cell-surface proteins regulate the immune system in humans. These proteins are present on the surface of cells, and they help the immune system distinguish foreign invaders such as viruses and bacteria from the body's own cells. Variants in the HLA genes are associated with more than 100 diseases, including infectious diseases like HIV, autoimmune conditions such as multiple sclerosis, and some cancers. However, while identifying which genetic variants are associated with an increased or decreased risk of disease is relatively simple, understanding why those genetic variants are associated with a particular disease is much harder. Debebe et al. have developed a new method to find out why certain gene variants in the HLA cluster are associated with disease in humans. They used this method to investigate known genetic variants associated with three viral infections: HIV, hepatitis C, and human leukemia virus ­ and one inflammatory disease: Crohn's disease. Critically, Debebe et al. looked at the interactions between different immune cells and the cell-surface proteins encoded by the HLA gene variants in different cases of these diseases. In doing so, the analysis was able to identify which cells of the immune system were responsible for the associations between gene variants and diseases. In principle, this method could be applied to study any disease in any species. It could also be used in classic gene association studies to test for false positive results and "passenger" mutations, two common problems that beset sound interpretations from these studies.

Inhibitory killer cell immunoglobulin-like receptors strengthen CD8+ T cell-mediated control of HIV-1, HCV, and HTLV-1.

Killer cell immunoglobulin-like receptors (KIRs) are expressed predominantly on natural killer cells, where they play a key role in the regulation of innate immune responses. Recent studies show that inhibitory KIRs can also affect adaptive T cell-mediated immunity. In mice and in human T cells in vitro, inhibitory KIR ligation enhanced CD8+ T cell survival. To investigate the clinical relevance of these observations, we conducted an extensive immunogenetic analysis of multiple independent cohorts of HIV-1-, hepatitis C virus (HCV)-, and human T cell leukemia virus type 1 (HTLV-1)-infected individuals in conjunction with in vitro assays of T cell survival, analysis of ex vivo KIR expression, and mathematical modeling of host-virus dynamics. Our data suggest that functional engagement of inhibitory KIRs enhances the CD8+ T cell response against HIV-1, HCV, and HTLV-1 and is a significant determinant of clinical outcome in all three viral infections.

The fate and lifespan of human monocyte subsets in steady state and systemic inflammation.

In humans, the monocyte pool comprises three subsets (classical, intermediate, and nonclassical) that circulate in dynamic equilibrium. The kinetics underlying their generation, differentiation, and disappearance are critical to understanding both steady-state homeostasis and inflammatory responses. Here, using human in vivo deuterium labeling, we demonstrate that classical monocytes emerge first from marrow, after a postmitotic interval of 1.6 d, and circulate for a day. Subsequent labeling of intermediate and nonclassical monocytes is consistent with a model of sequential transition. Intermediate and nonclassical monocytes have longer circulating lifespans (∼4 and ∼7 d, respectively). In a human experimental endotoxemia model, a transient but profound monocytopenia was observed; restoration of circulating monocytes was achieved by the early release of classical monocytes from bone marrow. The sequence of repopulation recapitulated the order of maturation in healthy homeostasis. This developmental relationship between monocyte subsets was verified by fate mapping grafted human classical monocytes into humanized mice, which were able to differentiate sequentially into intermediate and nonclassical cells.

3D Bayesian cluster analysis of super-resolution data reveals LAT recruitment to the T cell synapse.

Single-molecule localisation microscopy (SMLM) allows the localisation of fluorophores with a precision of 10-30 nm, revealing the cell's nanoscale architecture at the molecular level. Recently, SMLM has been extended to 3D, providing a unique insight into cellular machinery. Although cluster analysis techniques have been developed for 2D SMLM data sets, few have been applied to 3D. This lack of quantification tools can be explained by the relative novelty of imaging techniques such as interferometric photo-activated localisation microscopy (iPALM). Also, existing methods that could be extended to 3D SMLM are usually subject to user defined analysis parameters, which remains a major drawback. Here, we present a new open source cluster analysis method for 3D SMLM data, free of user definable parameters, relying on a model-based Bayesian approach which takes full account of the individual localisation precisions in all three dimensions. The accuracy and reliability of the method is validated using simulated data sets. This tool is then deployed on novel experimental data as a proof of concept, illustrating the recruitment of LAT to the T-cell immunological synapse in data acquired by iPALM providing ~10 nm isotropic resolution.

A Bayesian cluster analysis method for single-molecule localization microscopy data.

Cell function is regulated by the spatiotemporal organization of the signaling machinery, and a key facet of this is molecular clustering. Here, we present a protocol for the analysis of clustering in data generated by 2D single-molecule localization microscopy (SMLM)-for example, photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM). Three features of such data can cause standard cluster analysis approaches to be ineffective: (i) the data take the form of a list of points rather than a pixel array; (ii) there is a non-negligible unclustered background density of points that must be accounted for; and (iii) each localization has an associated uncertainty in regard to its position. These issues are overcome using a Bayesian, model-based approach. Many possible cluster configurations are proposed and scored against a generative model, which assumes Gaussian clusters overlaid on a completely spatially random (CSR) background, before every point is scrambled by its localization precision. We present the process of generating simulated and experimental data that are suitable to our algorithm, the analysis itself, and the extraction and interpretation of key cluster descriptors such as the number of clusters, cluster radii and the number of localizations per cluster. Variations in these descriptors can be interpreted as arising from changes in the organization of the cellular nanoarchitecture. The protocol requires no specific programming ability, and the processing time for one data set, typically containing 30 regions of interest, is ∼18 h; user input takes ∼1 h.

Human Stem Cell-like Memory T Cells Are Maintained in a State of Dynamic Flux.

Adaptive immunity requires the generation of memory T cells from naive precursors selected in the thymus. The key intermediaries in this process are stem cell-like memory T (TSCM) cells, multipotent progenitors that can both self-renew and replenish more differentiated subsets of memory T cells. In theory, antigen specificity within the TSCM pool may be imprinted statically as a function of largely dormant cells and/or retained dynamically by more transitory subpopulations. To explore the origins of immunological memory, we measured the turnover of TSCM cells in vivo using stable isotope labeling with heavy water. The data indicate that TSCM cells in both young and elderly subjects are maintained by ongoing proliferation. In line with this finding, TSCM cells displayed limited telomere length erosion coupled with high expression levels of active telomerase and Ki67. Collectively, these observations show that TSCM cells exist in a state of perpetual flux throughout the human lifespan.