How CD4⁺ T Cells Transcriptional Profile Is Affected by Culture Conditions: Towards the Design of Optimal In Vitro HIV Reactivation Assays

• Type of Material: Article
• Authors: Giuseppe Rubens Pascucci, IRCCS Ospedale Pediatrico Bambino Gesù; Elena Morrocchi, IRCCS Ospedale Pediatrico Bambino Gesù; Chiara Pighi, IRCCS Ospedale Pediatrico Bambino Gesù; Arianna Rotili, Università degli Studi di Roma "Tor Vergata"; Alessia Neri, IRCCS Ospedale Pediatrico Bambino Gesù; Chiara Medri, IRCCS Ospedale Pediatrico Bambino Gesù; Giulio Olivieri, IRCCS Ospedale Pediatrico Bambino Gesù; Marco Sanna, IRCCS Ospedale Pediatrico Bambino Gesù; Gianmarco Rasi, IRCCS Ospedale Pediatrico Bambino Gesù; Deborah Persaud, Johns Hopkins School of Medicine; Ann Chahroudi, Emory University; Mathias Lichterfeld, Massachusetts Institute of Technology; Eleni Nastouli, UCL Great Ormond Street Institute of Child Health; Caterina Cancrini, IRCCS Ospedale Pediatrico Bambino Gesù; Donato Amodio, IRCCS Ospedale Pediatrico Bambino Gesù; Paolo Rossi, IRCCS Ospedale Pediatrico Bambino Gesù; Nicola Cotugno, IRCCS Ospedale Pediatrico Bambino Gesù; Paolo Palma, IRCCS Ospedale Pediatrico Bambino Gesù
• Language: English
• Date: 2023-03-01
• Publisher: MDPI
• Publication Version: Final Published Version
• Copyright Statement: © 2023 by the authors.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.3390/biomedicines11030888
• Title of Journal or Parent Work: Biomedicines
• Volume: 11
• Issue: 3
• Grant/Funding Information: This work was supported by federal funds from the U.S. National Institutes of Health (NIH) through the Pediatric Adolescent Virus Elimination (PAVE) Martin Delaney Collaboratory Project Number 1UM1 AI164566-01 (https://www.pave-collaboratory.org/, accessed on 30 January 2023), and from grant U01 AI135941-04 to CP and EM.
• Keywords: in vitro cultures; RNA sequencing; Oxford Nanopore technologies; RPMI; PMA/ionomycin stimulation; autologous plasma; TCR signaling cascade; CD4+ T cells; T cell activation; HIV reactivation
• Research Categories: Health Sciences, Medicine and Surgery

Abstract

Most of the current assays directed at the investigation of HIV reactivation are based on cultures of infected cells such as Peripheral Blood Mononuclear Cells (PBMCs) or isolated CD4+ T cells, stimulated in vitro with different activator molecules. The culture media in these in vitro tests lack many age- and donor-specific immunomodulatory components normally found within the autologous plasma. This triggered our interest in understanding the impact that different matrices and cell types have on T cell transcriptional profiles following in vitro culture and stimulation. Methods: Unstimulated or stimulated CD4+ T cells of three young adults with perinatal HIV-infection were isolated from PBMCs before or after culture in RPMI medium or autologous plasma. Transcriptomes were sequenced using Oxford Nanopore technologies. Results: Transcriptional profiles revealed the activation of similar pathways upon stimulation in both media with a higher magnitude of TCR cascade activation in CD4+ lymphocytes cultured in RPMI. Conclusions: These results suggest that for studies aiming at quantifying the magnitude of biological mechanisms under T cell activation, the autologous plasma could better approximate the in vivo environment. Conversely, if the study aims at defining qualitative aspects, then RPMI culture could provide more evident results.