Biology subjects

Lukowski, S. W.

Publications and source records attributed to Lukowski, S. W..

4 recordsLinked to original sources

bioRxiv2024

T-cell receptors identified by a personalized antigen-agnostic screening approach target shared neoantigen KRAS Q61H

Adoptive cell therapy (ACT) with TCR-engineered T-cells represents a promising alternative to TIL- or CAR-T therapies for patients with advanced solid cancers. Currently, selection of therapeutic TCRs critically depends on knowing the target antigens, a condition excluding most patients from treatment. Direct antigen-agnostic identification of tumor-specific T-cell clonotypes and TCR-T manufacturing using their TCRs can advance ACT for patients with aggressive solid cancers. We present a method to identify tumor-specific clonotypes from surgical specimens by comparing TCR{beta}-chain repertoires of TILs and adjacent tissue-resident lymphocytes. In seven NSCLC-patients, tumor-specific clonotypes were selected based on TIL-abundance and high tumor-to-nontumor frequency ratios. In two of the patients, we demonstrate that predicted tumor-specific clonotypes reacted against autologous tumors. In a third patient, we engineered TCR T-cells with four candidate tumor-specific TCRs that showed reactivity against the patients tumor and HLA-matched NSCLC cell lines. The TCR-T cells were then used to screen for candidate neoantigens and aberrantly expressed antigens. Three TCRs recognized recurrent driver-mutation KRAS Q61H-peptide ILDTAGHEEY presented by HLA-A*01:01. The TCRs were also dominant in a tumor relapse, one was found in cell free DNA. The finding of homologous TCRs in independent KRAS Q61H-positive cancers suggests a therapeutic opportunity for HLA-matched patients with KRAS Q61H-expressing tumors.

immunology↗

bioRxiv2024

Iso-Seq enables discovery of novel isoform variants in human retina at single cell resolution

Recent single cell transcriptomic profiling of the human retina provided important insights into the genetic signals in heterogeneous retinal cell populations that enable vision. However, conventional single cell RNAseq with 3 short-read sequencing is not suitable to identify isoform variants. Here we utilized Iso-Seq with full-length sequencing to profile the human retina at single cell resolution for isoform discovery. We generated a retina transcriptome dataset consisting of 25,302 nuclei from three donor retina, and detected 49,710 known transcripts and 241,949 novel transcripts across major retinal cell types. We surveyed the use of alternative promoters to drive transcript variant expression, and showed that 1-8% of genes utilized multiple promoters across major retinal cell types. Also, our results enabled gene expression profiling of novel transcript variants for inherited retinal disease (IRD) genes, and identified differential usage of exon splicing in major retinal cell types. Altogether, we generated a human retina transcriptome dataset at single cell resolution with full-length sequencing. Our study highlighted the potential of Iso-Seq to map the isoform diversity in the human retina, providing an expanded view of the complex transcriptomic landscape in the retina.

genomics↗

bioRxiv2018

Longitudinal expression profiling of CD4+ and CD8+ cells in patients with active to quiescent Giant Cell Arteritis

BackgroundGiant cell arteritis (GCA) is the most common form of vasculitis affecting elderly people. It is one of the few true ophthalmic emergencies. GCA is a heterogenous disease, symptoms and signs are variable thereby making it challenging to diagnose and often delaying diagnosis. A temporal artery biopsy is the gold standard to test for GCA, and there are currently no specific biochemical markers to categorize or aid diagnosis of the disease. We aimed to identify a less invasive method to confirm the diagnosis of GCA, as well as to ascertain clinically relevant predictive biomarkers by studying the transcriptome of purified peripheral CD4+ and CD8+ T lymphocytes in patients with GCA.\n\nMethods and FindingsWe recruited 16 patients with histological evidence of GCA at the Royal Victorian Eye and Ear Hospital (RVEEH), Melbourne, Australia, and aimed to collect blood samples at six time points: acute phase, 2-3 weeks, 6-8 weeks, 3 months, 6 months and 12 months after clinical diagnosis. CD4+ and CD8+ T-cells were positively selected at each time point through magnetic-assisted cell sorting (MACS). RNA was extracted from all 195 collected samples for subsequent RNA sequencing. The expression profiles of patients were compared to those of 16 age-matched controls. Over the 12-month study period, polynomial modelling analyses identified 179 and 4 statistically significant transcripts with altered expression profiles (FDR < 0.05) between cases and controls in CD4+ and CD8+ populations, respectively. In CD8+ cells, we identified two transcripts that remained differentially expressed after 12 months, namely SGTB, associated with neuronal apoptosis, and FCGR3A, which has been found in association with Takayasu arteritis (TA), another large vessel vasculitis. We detected genes that correlate with both symptoms and biochemical markers used in the acute setting for predicting long-term prognosis. 15 genes were shared across 3 phenotypes in CD4 and 16 across CD8 cells. In CD8, IL32 was common to 5 phenotypes: a history of Polymyalgia Rheumatica, both visual disturbance and raised neutrophils at the time of presentation, bilateral blindness and death within 12 months. Altered IL32 gene expression could provide risk evaluation of GCA diagnosis at the time of presentation and give an indication of prognosis, which may influence management.\n\nConclusionsThis is the first longitudinal gene expression study undertaken to identify robust transcriptomic biomarkers of GCA. Our results show cell type-specific transcript expression profiles, novel gene-phenotype associations, and uncover important biological pathways for this disease. These data significantly enhance the current knowledge of relevant biomarkers, their association with clinical prognostic markers, as well as potential candidates for detecting disease activity in whole blood samples. In the acute phase, the gene-phenotype relationships we have identified could provide insight to potential disease severity and as such guide us in initiating appropriate patient management.

genomics↗

bioRxiv2017

Single Cell RNA Sequencing of stem cell-derived retinal ganglion cells.

We used human embryonic stem cell-derived retinal ganglion cells (RGCs) to characterize the transcriptome of 1,174 cells at the single cell level. The human embryonic stem cell line BRN3B-mCherry A81-H7 was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and subsequently prepared for single cell RNA sequencing. Our data indicates the presence of three distinct subpopulations of cells, with various degrees of maturity. One cluster of 288 cells upregulated genes involved in axon guidance together with semaphorin interactions, cell-extracellular matrix interactions and ECM proteoglycans, suggestive of a more mature phenotype.

genomics↗