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The EasySep™ Human Resting CD4+ T Cell Isolation Kit is designed to isolate resting CD4+ T cells from fresh or previously frozen human peripheral blood mononuclear cells or washed leukapheresis samples by immunomagnetic negative selection. The EasySep™ procedure involves labeling unwanted cells with antibody complexes and magnetic particles. The magnetically labeled cells are separated from the untouched desired cells by using an EasySep™ magnet and simply pouring or pipetting the desired cells into a new tube.
(A) Starting with fresh PBMCs, the resting CD4+ T cell content (CD3+CD4+CD8-CD25-CD69-HLA-DR-) of the isolated fraction is typically 95.5 ± 6.5% (mean ± SD using the purple EasySep™ Magnet). In the above example, the purities of the start and final isolated fractions are 35% and 99%, respectively. (B) Starting with fresh PBMCs that have elevated CD25/CD69/HLA-DR expression, the resting CD4+ T cell content of the isolated fraction is typically 88.0 ± 7.0% (mean ± SD using the purple EasySep™ Magnet). In the above example, the purities of the start and final isolated fractions are 8% and 85%, respectively.
This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.
Endothelial Cells Promote Productive HIV Infection of Resting CD4+ T Cells by an Integrin-Mediated Cell Adhesion-Dependent Mechanism. C. M. Card et al. AIDS research and human retroviruses 2022 feb
Abstract
Resting CD4+ T cells are primary targets of early HIV infection events in vivo, but do not readily support HIV replication in vitro. This barrier to infection can be overcome by exposing resting CD4+ T cells to endothelial cells (ECs). ECs line blood vessels and direct T cell trafficking into inflamed tissues. Cell trafficking pathways have been shown to have overlapping roles in facilitating HIV replication, but their relevance to EC-mediated enhancement of HIV susceptibility in resting CD4+ T cells has not previously been examined. We characterized the phenotype of primary human resting CD4+ T cells that became productively infected with HIV when cocultured with primary human blood and lymphatic ECs. The infected CD4+ T cells were primarily central memory cells enriched for high expression of the integrins LFA-1 and VLA-4. ICAM-1 and VCAM-1, the cognate ligands for LFA-1 and VLA-4, respectively, were expressed by the ECs in the coculture. Blocking LFA-1 and VLA-4 on resting CD4+ T cells inhibited infection by 65.4%-96.9%, indicating that engagement of these integrins facilitates EC-mediated enhancement of productive HIV infection in resting CD4+ T cells. The demonstration that ECs influence cellular HIV susceptibility of resting memory CD4+ T cells through cell trafficking pathways engaged during the transmigration of T cells into tissues highlights the physiological relevance of these findings for HIV acquisition and opportunities for intervention.
Hepatitis C Virus Influences HIV-1 Viral Splicing in Coinfected Patients. P. Mart\'inez-Rom\'an et al. Journal of clinical medicine 2020 jul
Abstract
Coinfection with hepatitis C virus (HCV) influences HIV reservoir size. However, it is unknown whether this coinfection also induces a higher provirus transcription. Viral transcription is promoted by synergy between cellular factors such as NF-$\kappa$B and the viral regulator Tat. The impact of HCV coinfection on HIV provirus transcription was analyzed in resting (r)CD4 T+ cells (CD3+CD4+CD25-CD69-HLADR-) and rCD4 T cells-depleted PBMCs (rCD4 T- PBMCs) from a multicenter cross-sectional study of 115 cART-treated HIV patients: 42 HIV+/HCV+ coinfected individuals (HIV+/HCV+), 34 HIV+ patients with HCV spontaneous clearance (HIV+/HCV-) and 39 HIV patients (HIV+). Viral transcription was assessed in total RNA through the quantification of unspliced, single spliced, and multiple spliced viral mRNAs by qPCR. Linear correlations between viral reservoir size and viral splicing were determined. A 3-fold increase of multiple spliced transcripts in rCD4 T+ cells of HIV+/HCV+ patients was found compared to HIV+ individuals (p {\textless} 0.05). As Tat is synthesized by multiple splicing, the levels of Tat were also quantified in these patients. Significant differences in single and multiple spliced transcripts were also observed in rCD4 T- PBMCs. Levels of multiple spliced mRNAs were increased in rCD4 T+ cells isolated from HIV+/HCV+ subjects, which could indicate a higher Tat activity in these cells despite their resting state.
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