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

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.