An In vitro Model for the Study of HIV Infection of Thymocytes

Abstract

The study of human thymocytes requires an appropriate matrix to enable the proper function of thymocytes. Although OP9-DL1 cells have been well established as an ideal co-culture system for the generation of T-cells from their progenitors; their ability to support a mixed population of mature human thymocytes for in vitro HIV infection studies has yet to be established. We assessed the effects of co-culturing a heterogeneous population of mature human thymocytes with a mouse derived cell line (OP9) transduced with the notch ligand delta like 1 (OP9-DL-1) and compared this to standard co-culture with human thymic epithelial cells (TEC). Co-culturing thymocytes with OP9-DL1 cells resulted in higher viability and lower apoptosis when compared to TEC co-cultures. The subset distribution and CD127 expression of thymocytes varied slightly between conditions. Thymocytes co-cultured with OP9-DL1 cells had a lower proportion of CD3 + DP cells and higher proportion of SP4 cells compared to TEC co-cultures. The mature CD3 + CD4 + CD8-(SP4) cells also had lower levels of CD127 expression in OP9-DL1 cultures when compared to TEC. Interleukin-7 stimulation of thymocytes resulted in a decrease in CD127 expression in OP9-DL1 co-cultures, as previously observed with TEC co-cultures. Thymocytes co-cultured with OP9-DL1 tended to have higher levels of IL-7 induced STAT-5 phosphorylation and had higher levels of Interleukin-7 induced Bcl-2 expression. OP9-DL1 cells provide a microenvironment which is permissive to HIV infection in thymocytes in vitro. Co-culturing thymocytes with OP9-DL1 will facilitate the study of human thymocytes and aid in the study of exogenous stimuli or infection on individual thymocyte subsets.

A Review on Various Aspects of HIV Infection

“…It was being clearly stated that co-culture of human thymocytes with OP9-DL1 cells result in greater IL-7 response when compared to co-cultures with thymic epithelial cells. This reveals that they are an appropriate candidate for exogenous stimuli on thymocyte function [37].…”

Impact of in vitro HIV infection on human thymic regulatory T cell differentiation

“…One of the main reasons for using OP9-DL1 co-culture, instead of the in vitro stimulation of thymocytes via anti-CD3 or cytokines such as IL-2, is that such stimulations either increase FoxP3 and/or CD25 expression on CD4 T cells ( Sereti et al, 2000 ; Wang et al, 2007 ). Furthermore, our team previously reported this system as an optimal model to study the differentiation of human thymocytes in vitro , as well as their exogenous stimulation or HIV infection ( Young and Angel, 2011 ; Young, 2012 ). Our in vitro data indicate that tTregs are refractory to HIV infection, which could be related to the fact that the transcription factor FoxP3 may block HIV viral transcription by restraining the expression of NFAT ( Selliah et al, 2008 ).…”

“…In order to avoid the impact of TCR stimulation on the expression of FOXP3, all in vitro stimulation of human thymocytes and HIV infection experiments were carried out using a co-culture model with the mouse stromal cell line OP9-DL1 – which expresses the ligand for Notch, delta-like-1 (DL-1) as we previously reported ( Young and Angel, 2012 ). Notch signaling is essential for T cell differentiation and lineage commitment in the thymus.…”

“…This cell line was generously provided by Dr. Zúñiga-Pflücker, University of Toronto. The co-culture has been established with a ratio of thymocyte: OP9- DL1 of 25:1 ( Young and Angel, 2012 ). OP9-DL1 cells were maintained in culture in AMEM (Wisent) containing 20% FBS and 1% penicillin–streptomycin and passaged every 2–3 days using trypsin–EDTA (Wisent).…”

“…Our team has previously reported that OP9- DL1 co-culture is an optimal model for in vitro stimulation and HIV infection ( Young and Angel, 2011 ; Young and Angel, 2012 ). One million of freshly isolated or thawed thymocytes were treated with polybrene (Sigma Aldrich, Canada) at 3 μg per 10 6 cells for 1 h, at 37°C to increase the infection rate.…”