Microfluidic Nucleic Acid Cytometry Reveals Transcriptional Programs of HIV Silencing and Cell Survival in HIV Infected Memory CD4 T Cells
Rare CD4 T cells that harbor HIV under antiretroviral therapy (ART) represent an important barrier to HIV cure. However, the infeasibility of isolating and characterizing these cells has severely limited our understanding of the cellular mechanisms that control their fate and has led to uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit. Major technical obstacles have prevented characterization of the cellular reservoir in people on ART. First, HIV-infected cells are exceedingly rare, accounting for less than 0.1% of the CD4 T cell pool. Second, they contain no known markers that might be exploited for their enrichment and characterization. Finally, they are widely distributed across T cell subsets, suggesting that defining how HIV infected cells differ from their uninfected counterparts will require an unbiased “omic” approach. To overcome these challenges, we developed FIND-seq (Focused Interrogation of cells by Nucleic acid Detection and Sequencing), a high-throughput cytometry method that isolates cells based on single-copy DNA markers. FIND-Seq uses microfluidic devices to perform cell capture, purification, and transcriptome processing on millions of cells in parallel. FIND-seq can access the unique biology of a broad array of previously unsortable cell types including rare cell subsets defined by unique gene expression signatures or cells containing novel genetic features, including mutations or integrated viruses. Using FIND-seq, we profiled HIV-infected CD4 T cells from six people living with HIV, revealing that these cells are a highly selected population, maintained in vivo through a diverse but definable array of mechanisms. Specifically, HIV infected cells express transcriptional programs that favor proliferation, HIV silencing, and resistance to cell death. Our results unify and extend decades of observations made from in vitro experiments and elucidate new genes and pathways that may control HIV persistence in vivo.
Bio: Iain C. Clark, Ph.D. is an Assistant Professor in the Bioengineering Department at the University of California, Berkeley and a faculty affiliate at the California Institute for Quantitative Biosciences. He received his BS in Biological Engineering from Cornell University, an MS in Biosystems Engineering from UC Davis, and a PhD in Environmental Engineering from UC Berkeley. As postdoc at UCSF, he used fluid mechanics, computer modeling, and microfabrication to advance droplet microfluidic technologies for single cell analysis. His work led to the development of multiple new microfluidic devices and enabled ultra-high throughput and integrated on-chip cell processing, targeted single cell barcoding, and single cell genome and transcriptome sequencing. As a fellow at Harvard Medical School, he pioneered new methods for isolating and studying rare pathologic cells and developed novel barcoded viral tracing methods for brain connectomics. His work has been published in top microfluidic and biology journals, including Nature and Science, and he is a recipient of a NIH K22 Career Transition Award and the NIAID New Innovator Award. Research in the Clark lab at UC Berkeley combines tools from multiple disciplines - molecular biology, microfluidics, engineering, and bioinformatics - to study infectious diseases, central nervous system inflammation, and neural circuit connectivity. Current projects center on two biological questions with important human health implications: what are the unique properties of cells harboring latent HIV in vivo, and what are the molecular mechanisms that control cell interactions in neurodegenerative diseases like multiple sclerosis and Alzheimer's disease?