Benn, C. | Search bioRxiv

bioRxiv2025

Spatiotemporal-multimodal integration reveals BCG-induced skin-blood crosstalk

Tuberculosis (TB) remains the leading cause of infectious death. The Bacille Calmette-Guerin (BCG) vaccine has been the only licensed vaccine available for TB prevention. Despite BCG being administered intradermally for over a century to >100 million individuals annually, the molecular events in the skin following BCG administration have not been investigated; as a result, measurable correlates of protection that could predict vaccine effectiveness already early after vaccination are lacking. Here we show that BCG immediately (within one day after vaccination) induces dynamic molecular waves that drive the acute human host response across space (layers of the skin and systemically in blood) and time (days). Integration of this data across space and time identified robust networks of interactive modules related to immune surveillance (e.g. Langerhans cells), cell trafficking (e.g. endothelial cells, ITGB5), and trained immunity (e.g. neutrophils, macrophages, {gamma}{delta}-T cell). Importantly, not only were we able to identify BCG-activated pathways associated with trained immunity such as mTOR signaling and glycolysis/gluconeogenesis, we were able to pinpoint the time-point and precise location (skin layer) of the initial activation of theses pathways. Combining tissue biopsies of human skin (spatial genomics) with liquid biopsies (cell-free blood plasma RNASeq) following BCG vaccination our data both confirmed known evidence (e.g. prominent {gamma}{delta}-T cell induction at the site of BCG administration; negative correlation of blood vs tissue myeloid-derived suppressor cells), but also generated promising new leads such as baseline levels of B cells, platelets and nuocytes in the skin prior to BCG administration predict eventual outcome, and that these predictive differences in baseline cellular composition can be captured non-invasively using high resolution images of the site of injection (dermatoscopy). Given this data represents the first holistic view of the acute molecular response to BCG in the skin in a human population at medium to high TB risk, we anticipate our findings of the immediate/early events following BCG vaccination, including non-invasive predictive assessment will support acceleration of vaccine development in the fight against TB.

systems biology↗

bioRxiv2024

The TRX assay for triplet repeat expansions

Expansion mutations of triplet repeat sequences cause numerous inherited neurological diseases. In some diseases, affected individuals display somatic expansions in affected tissues which have been linked to accelerated disease onset and progression. There is currently considerable interest in developing therapies to slow somatic repeat expansions to delay or block disease onset. In vitro assays are particularly important to evaluate potential therapeutic interventions. Current assays typically use physical methods to monitor triplet repeat lengths within a population of cells. While useful, most of these assays are relatively slow ([~]six weeks) and are somewhat limited in sensitivity to rare events. Here, a new assay, called TRX, is described to monitor CAG*CTG triplet repeat expansions more rapidly and with better sensitivity. TRX uses human tissue culture cells expressing two fluorescent proteins. Red fluorescent protein TagRFP658 is constitutively expressed and serves as an internal control. GFP is expressed in a CAG*CTG repeat length-dependent manner, with longer repeat lengths predicted to give higher green fluorescence intensity. Standard flow cytometry allows quantification of changes in fluorescent signal as a simple readout with <2% sensitivity. Two independently derived cell lines with 63 or 59 CAG repeats yielded similar rates of TRX activity. Cells with increased green fluorescence were observed within one to two weeks of culture, with longer times leading to additional signal. The appearance of green fluorescence was partly dependent on MutS{beta}, the DNA MSH2-MSH3 complex, based on siRNA knockdown of MSH3. However, physical analysis of the CAG*CTG repeat tracts by MiSeq deep sequencing or capillary electrophoresis showed limited changes in the length of the repeat tracts. We conclude that the TRX assay is a promising new tool for monitoring CAG*CTG repeat expansions but that further development of the assay is needed to make it fully useful.

genetics↗