Automated microarray platform for single-cell sorting and collection of lymphocytes following HIV reactivation.

Autor: Cortés-Llanos B; Department of Bioengineering University of Washington Washington USA.; Department of Medicine Duke University North Carolina USA., Jain V; Department of Molecular Physiology Duke University North Carolina USA., Cooper-Volkheimer A; Department of Medicine Duke University North Carolina USA., Browne EP; Department of Medicine University of North Carolina North Carolina USA.; Department of Microbiology and Immunology University of North Carolina North Carolina USA.; UNC HIV Cure Center University of North Carolina North Carolina USA., Murdoch DM; Department of Medicine Duke University North Carolina USA., Allbritton NL; Department of Bioengineering University of Washington Washington USA.
Jazyk: angličtina
Zdroj: Bioengineering & translational medicine [Bioeng Transl Med] 2023 Jun 21; Vol. 8 (5), pp. e10551. Date of Electronic Publication: 2023 Jun 21 (Print Publication: 2023).
DOI: 10.1002/btm2.10551
Abstrakt: A promising strategy to cure HIV-infected individuals is to use latency reversing agents (LRAs) to reactivate latent viruses, followed by host clearance of infected reservoir cells. However, reactivation of latent proviruses within infected cells is heterogeneous and often incomplete. This fact limits strategies to cure HIV which may require complete elimination of viable virus from all cellular reservoirs. For this reason, understanding the mechanism(s) of reactivation of HIV within cellular reservoirs is critical to achieve therapeutic success. Methodologies enabling temporal tracking of single cells as they reactivate followed by sorting and molecular analysis of those cells are urgently needed. To this end, microraft arrays were adapted to image T-lymphocytes expressing mCherry under the control of the HIV long terminal repeat (LTR) promoter, in response to the application of LRAs (prostratin, iBET151, and SAHA). In response to prostratin, iBET151, and SAHA, 30.5%, 11.2%, and 12.1% percentage of cells, respectively. The arrays enabled large numbers of single cells (>25,000) to be imaged over time. mCherry fluorescence quantification identified cell subpopulations with differing reactivation kinetics. Significant heterogeneity was observed at the single-cell level between different LRAs in terms of time to reactivation, rate of mCherry fluorescence increase upon reactivation, and peak fluorescence attained. In response to prostratin, subpopulations of T lymphocytes with slow and fast reactivation kinetics were identified. Single T-lymphocytes that were either fast or slow reactivators were sorted, and single-cell RNA-sequencing was performed. Different genes associated with inflammation, immune activation, and cellular and viral transcription factors were found.
Competing Interests: Nancy L. Allbritton discloses a financial interest in cell microsystems, Inc. All other authors declare no conflicts.
(© 2023 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of The American Institute of Chemical Engineers.)
Databáze: MEDLINE
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