Impact of media culture and PBMCs signaling on in vitro CD4+ T cells cultures to an optional HIV reactivation assay design.

Most of the current assays directed at the investigation of HIV reactivation are based on cultures of infected cells stimulated in vitro with different activatory molecules (i.e.TILDA and QVOA test). The RPMI culture media developed for these tests often include animal components (i.e. Fetal bovine serum, FBS) for their practicality and affordability, but nonetheless lack many age- and donor-specific immunomodulatory components normally found within the autologous plasma. This triggered our interest in understanding the possible impact that different matrices (RPMI or autologous plasma) and the other PBMCs could have on T cell expression profiles, collected from people living with HIV, when cultured/stimulated in vitro. Transcriptional profiles of CD4+ T cells isolated before or after in vitro cell culture from three ART-treated young adults with HIV perinatal infection, highlighted a mitigatory action of plasma on CD4 lymphocytes upon stimulation. Overall design: We explored the full transcriptome of CD4+ T cells from 3 HIV donors evaluating the impact of isolating the CD4 pre-culture (PREc) or post-culture (POSTc), using autologous plasma (PLSM) or RPMI as culture matrix. Cells were stimulated for 18 h with PMA/IONO and IL-2 together with AZT, or left unstimulated for both matrices (Fig.1). Taking into account all these variables, we analyzed 8 different experimental condition combinations for each donor, plus a sample of un-cultured CD4+ T cells (T0). Overall, a total of 27 samples were sequenced using the MiniION Oxford Nanopore platform.