Cells were stained by standard protocols as described previously.[15 (link),16 (link)] The following antibodies were used to define T cell subsets (CD3, CD4, CD8, γδTCR), T cell activation (CD38, HLA-DR, CCR5, CD69, PD-1), and T cell differentiation into naïve and memory T cells, including tissue resident memory cells, (CCR7, CD45RA, CD69). The analysis of γδ T cells was included because γδ T cells are frequently found at mucosal surfaces, are rapidly depleted in HIV infection, and have also been demonstrated to serve as a viral reservoir for HIV.[17 (link),18 (link)] All antibodies were obtained from BD Biosciences (San Jose, CA), except for PD-1 that was obtained from eBioscience (San Diego, CA). A live/dead cell discriminator dye (Qdot 585; Invitrogen, ThermoFisher Scientific; Waltham, MA) was included in all samples. Samples were acquired on a LSRII instrument (Beckton Dickinson, San Jose, CA) recording a minimum of 300,000 events. Samples were analyzed with FlowJo Software (Treestar, Ashland, OR) applying Boolean gating strategies.
Protocol detail
Comprehensive T Cell Profiling in HIV Infection
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A 300
Antibodies
Ccr5
Cell
Cell differentiation
Hiv infection
Hla dr
Memory
Memory t cells
Mucosal
T cell subsets
T cells
Tissue
Corresponding Organization : University of North Carolina at Chapel Hill
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Variable analysis
independent variables
- Cell staining
- Antibodies used to define T cell subsets, T cell activation, and T cell differentiation
- T cell subsets (CD3, CD4, CD8, γδTCR)
- T cell activation (CD38, HLA-DR, CCR5, CD69, PD-1)
- T cell differentiation into naïve and memory T cells, including tissue resident memory cells (CCR7, CD45RA, CD69)
- Frequency and function of γδ T cells
- Use of a live/dead cell discriminator dye (Qdot 585)
- Minimum of 300,000 events recorded during sample acquisition on a LSRII instrument
- Not explicitly mentioned.
- Not explicitly mentioned.
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