Aridaman Pandit

Reliable reconstruction of HIV-1 whole genome haplotypes reveals clonal interference and genetic hitchhiking among immune escape variants

BackgroundFollowing transmission, HIV-1 evolves into a diverse population, and next generation sequencing enables us to detect variants occurring at low frequencies. Studying viral evolution at the level of whole genomes was hitherto not possible because next generation sequencing delivers relatively short reads.ResultsWe here provide a proof of principle that whole HIV-1 genomes can be reliably reconstructed from short reads, and use this to study the selection of immune escape mutations at the level of whole genome haplotypes. Using realistically simulated HIV-1 populations, we demonstrate that reconstruction of complete genome haplotypes is feasible with high fidelity. We do not reconstruct all genetically distinct genomes, but each reconstructed haplotype represents one or more of the quasispecies in the HIV-1 population. We then reconstruct 30 whole genome haplotypes from published short sequence reads sampled longitudinally from a single HIV-1 infected patient. We confirm the reliability of the reconstruction by validating our predicted haplotype genes with single genome amplification sequences, and by comparing haplotype frequencies with observed epitope escape frequencies.ConclusionsPhylogenetic analysis shows that the HIV-1 population undergoes selection driven evolution, with successive replacement of the viral population by novel dominant strains. We demonstrate that immune escape mutants evolve in a dependent manner with various mutations hitchhiking along with others. As a consequence of this clonal interference, selection coefficients have to be estimated for complete haplotypes and not for individual immune escapes.

RTCR: a pipeline for complete and accurate recovery of T cell repertoires from high throughput sequencing data

Motivation: High Throughput Sequencing (HTS) has enabled researchers to probe the human T cell receptor (TCR) repertoire, which consists of many rare sequences. Distinguishing between true but rare TCR sequences and variants generated by polymerase chain reaction (PCR) and sequencing errors remains a formidable challenge. The conventional approach to handle errors is to remove low quality reads, and/or rare TCR sequences. Such filtering discards a large number of true and often rare TCR sequences. However, accurate identification and quantification of rare TCR sequences is essential for repertoire diversity estimation. Results: We devised a pipeline, called Recover TCR (RTCR), that accurately recovers TCR sequences, including rare TCR sequences, from HTS data (including barcoded data) even at low coverage. RTCR employs a data-driven statistical model to rectify PCR and sequencing errors in an adaptive manner. Using simulations, we demonstrate that RTCR can easily adapt to the error profiles of different types of sequencers and exhibits consistently high recall and high precision even at low coverages where other pipelines perform poorly. Using published real data, we show that RTCR accurately resolves sequencing errors and outperforms all other pipelines. Availability and Implementation: The RTCR pipeline is implemented in Python (v2.7) and C and is freely available at http://uubram.github.io/RTCR/along with documentation and examples of typical usage. Contact: b.gerritsen@uu.nl

The memory of a killer T cell: models of CD8 + T cell differentiation

CD8+ T cells have an important role in protection against infections and reinfections of intra‐cellular pathogens like viruses. Naive CD8+ T cells circulating in blood or lymphoid tissues can get activated upon stimulation by cognate antigen. The activated T cells undergo rapid proliferation and can expand more than 104‐folds comprising largely of effector T cells. Upon antigen clearance, the CD8+ T‐cell population contracts due to apoptosis, leaving behind a small population of memory T cells. The timing and mechanisms underlying the differentiation of naive cells into effector cells and memory cells is not yet clear. In this article, we review the recent quantitative studies that support different hypotheses of CD8+ T‐cell differentiation.

Is sex bias orchestrated in the skin

The identification of VGLL3 as a transcription (co-)factor that underlies the sex bias of the human immune system further underscores the relevance of research into this area.

HIV-1 CCR5 gene therapy will fail unless it is combined with a suicide gene

Highly active antiretroviral therapy (ART) has successfully turned Human immunodeficiency virus type 1 (HIV-1) from a deadly pathogen into a manageable chronic infection. ART is a lifelong therapy which is both expensive and toxic, and HIV can become resistant to it. An alternative to lifelong ART is gene therapy that targets the CCR5 co-receptor and creates a population of genetically modified host cells that are less susceptible to viral infection. With generic mathematical models we show that gene therapy that only targets the CCR5 co-receptor fails to suppress HIV-1 (which is in agreement with current data). We predict that the same gene therapy can be markedly improved if it is combined with a suicide gene that is only expressed upon HIV-1 infection.

A Disease-Associated MicroRNA Cluster Links Inflammatory Pathways and an Altered Composition of Leukocyte Subsets to Noninfectious Uveitis

Purpose The cause of noninfectious uveitis (NIU) is poorly understood but is considered to be mediated by a complex interplay between genetic, environmental, and-relatively unexplored-epigenetic factors. MicroRNAs (miRNAs) are noncoding small RNAs that are important epigenetic regulators implicated in pathologic signaling. Therefore, we mapped the circulating miRNA-ome of NIU patients and studied miRNA perturbations within the broader context of the immune system. Methods We designed a strategy to robustly identify changes in the miRNA profiles of two independent cohorts totaling 54 untreated patients with active and eye-restricted disease and 26 age-matched controls. High-resolution miRNA-ome data were obtained by TaqMan OpenArray technology and subsequent RT-qPCR. Flow cytometry data, and proteomic data spanning the cellular immune system, were used to map the uveitis-miRNA signature to changes in the composition of specific leukocyte subsets in blood. Results Using stringent selection criteria, we identified and independently validated an miRNA cluster that is associated with NIU. Pathway enrichment analysis for genes targeted by this cluster revealed significant enrichment for the PI3K/Akt, MAPK, FOXO, and VEGF signaling pathways, and photoreceptor development. In addition, unsupervised multidomain analyses linked the presence of the uveitis-associated miRNA cluster to a different composition of leukocyte subsets, more specifically, CD16+CD11c+HLA-DR- cells. Conclusions Together, this study identified a unique miRNA cluster associated with NIU that was related to changes in leukocyte subsets demonstrating systemic changes in epigenetic regulation underlying NIU.

Analysis of dinucleotide signatures in HIV-1 subtype B genomes

Dinucleotide usage is known to vary in the genomes of organisms. The dinucleotide usage profiles or genome signatures are similar for sequence samples taken from the same genome, but are different for taxonomically distant species. This concept of genome signatures has been used to study several organisms including viruses, to elucidate the signatures of evolutionary processes at the genome level. Genome signatures assume greater importance in the case of host–pathogen interactions, where molecular interactions between the two species take place continuously, and can influence their genomic composition. In this study, analyses of whole genome sequences of the HIV-1 subtype B, a retrovirus that caused global pandemic of AIDS, have been carried out to analyse the variation in genome signatures of the virus from 1983 to 2007. We show statistically significant temporal variations in some dinucleotide patterns highlighting the selective evolution of the dinucleotide profiles of HIV-1 subtype B, possibly a consequence of host specific selection.

microRNA downregulation in plasmacytoid dendritic cells in interferon-positive systemic lupus erythematosus and antiphospholipid syndrome

Objective To investigate miRNA expression in relation to transcriptomic changes in plasmacytoid dendritic cells (pDCs) in SLE and APS. pDCs are major producers of IFNα in SLE and APS, and miRNAs are emerging as regulators of pDC activation. Methods miRNA and mRNA expression were measured by OpenArray and RNA-sequencing in pDCs of SLE, SLE + APS (APS secondary to SLE) and primary APS (PAPS) patients. The miRNA profile of patients was compared with the miRNA pattern of TLR7-activated pDCs. Results Among 131 miRNAs detected in pDCs, 35, 17 and 21 had a significantly lower level of expression in SLE, SLE + APS and PAPS patients, respectively, as compared with healthy controls (HC). Notably, the miRNA profile did not significantly differ between SLE and APS, but was driven by the presence or absence of an IFN signature. TLR7 stimulation induced a general downregulation of miRNAs, similar to the pattern observed in SLE and APS patients. miR-361-5p, miR-128-3p and miR-181a-2-3p expression was lower in patients with a high IFN signature (false discovery rate <0.05) as compared with patients with a low IFN signature and HCs. Pathway enrichment on the overlap of miRNA targets and upregulated genes from the RNAseq indicated that these miRNAs are involved in pDC activation and apoptosis. Conclusion Lower miRNA expression in pDCs is shared between SLE, SLE + APS and PAPS and is related to the IFN signature. As pDCs are the alleged source of the IFN signature in these patients, a better understanding of the molecular mechanisms/pathways leading to pDC dysregulation in SLE and APS might open novel pathways for therapeutic intervention.

PS5:91 Increased expression of tlr7 in plasmacytoid dendritic cells drives type i ifn mediated immunopathology in sle and aps

Background/purpose Dendritic cells (DC) are key cells in the pathogenesis of autoimmune diseases by potently activating T-cells. Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are characterised by an IFN signature, caused by elevated levels of IFNα. Plasmacytoid DC (pDC) are held responsible for the increased levels of IFNα in SLE and APS. The molecular mechanisms underlying the increased activation of pDC in SLE and APS are unknown. Using RNA sequencing (RNAseq) and further in vitro validation experiments on plasmacytoid and myeloid DC (mDC) obtained from patients with SLE and APS we assessed the causes and consequences of increased type I IFN signalling on the dysregulation of both pDC and mDC in patients with SLE and APS. Methods RNAseq was performed on pDC and mDC isolated from peripheral blood of patients with SLE, SLE +APS and primary APS (PAPS) and healthy controls (n=54). Weighted gene correlation network analysis (WGCNA) was used to identify pDC- and mDC-specific gene modules and to stratify patients into those with (IFN-high) or without (IFN-low) an IFN signature. The response of pDC and mDC (in co-culture with T-cells) to IFNα and TLR agonists were analysed by RT-qPCR and flow cytometry to functionally validate RNAseq data. Results WGCNA identified IFN modules in pDC and mDC that perfectly stratified patients from HC. Comparing the IFN modules of pDC and mDC revealed cell specific alterations related to the IFN signature in pDC/mDC. Increased expression of TLR7 and its downstream intermediates was confined to IFN-high patients in pDC. In contrast, genes involved in the activation of T-cells were related to the IFN module in mDC. Both pDC and mDC showed increased expression of BAFF. In vitro, IFNα upregulated TLR7 in pDC and augmented TLR7-mediated IFNα production. In contrast to pDC, IFNα primed mDC for enhanced T-cell proliferation via the upregulation of co-stimulatory molecules. Furthermore, in SLE/APS, pDC and mDC produced BAFF and expressed chemokine receptors. Conclusion pDC and mDC are differentially affected by IFNα in SLE and APS. IFNα primes pDC for enhanced IFNα production which potentiates T-cell activation by mDC, thereby sustaining the IFN signature in SLE and APS.

High-dimensional profiling reveals heterogeneity of the Th17 subset and its association with systemic immunomodulatory treatment in non-infectious uveitis

Background: Non-infectious uveitis (NIU) is a severe intra ocular inflammation, which frequently requires prompt systemic immunosuppressive therapy (IMT) to halt the development of vision-threatening complications. IMT is considered when NIU cannot be treated with corticosteroids alone, which is unpredictable in advance. Previous studies have linked blood cell subsets to glucocorticoid sensitivity, which suggests that the composition of blood leukocytes may early identify patients that will require IMT. Objective: To map the blood leukocyte composition of NIU and identify cell subsets that stratify patients that required IMT during follow-up. Methods: We performed controlled flow cytometry experiments measuring a total of 37 protein markers in the blood of 30 IMT free patients with active non-infectious anterior, intermediate, and posterior uveitis, and compared these to 15 age and sex matched healthy controls. Results from manual gating were validated by automatic unsupervised gating using FlowSOM. Results: Patients with uveitis displayed lower relative frequencies of Natural Killer cells and higher relative frequencies of memory T cells, in particular the CCR6+ lineages. These results were confirmed by automatic gating by unsupervised clustering using FlowSOM. We observed considerable heterogeneity in memory T cell subsets and abundance of CXCR3-CCR6+ (Th17) cells between the uveitis subtypes. Importantly, regardless of the uveitis subtype, patients that eventually required IMT in the course of the study follow-up exhibited increased CCR6+ T cell abundance before commencing therapy. Conclusion: High-dimensional immunoprofiling in NIU patients shows that clinically distinct forms of human NIU exhibit shared as well as unique immune cell perturbations in the peripheral blood and link CCR6+ T cell abundance to systemic immunomodulatory treatment.