Computational protocol: Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover

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Protocol publication

[…] HLA-A*0301 QK10, HLA-A*0301 RY10, HLA-B*0702 GL9, and HLA-B*2705 KK10 pentamers were purchased from ProImmune; HLA-A*0201 SL9 tetramer was purchased from Beckman Coulter; and HLA-B*0801 FL9 tetramer was synthesized as previously described (). PBMCs were stained with pretitrated concentrations of pentamer/tetramer (conjugated to PE or allophycocyanin [APC]) followed by a panel of antibodies, as previously described (). Directly conjugated and unconjugated antibodies were as follows: CD3 (Cy5.5PerCP or Alexa Fluor 700), CD38 (APC), CD45RA (FITC), CCR7 (PE-Cy7), CD107a (Cy5-PE), IL-2 (APC), IFN-γ (Alexa Fluor 700), CD57 (FITC), and TNF-α (PE-Cy7; all from BD Biosciences); CD28 (PE–Texas red), CD45RA (PE–Texas red), TCRBV mAbs (5.1, 6.6, 9, 14, 13, and 20.1 conjugated to FITC or PE; all from Beckman Coulter); CD8 (Alexa Fluor 405), swine anti–goat (FITC), and granzyme B (PE–Texas red; all from Caltag); CD127 (purified) and MIP-1β (FITC; both from R&D Systems); PD-1 (PE; eBioscience); goat anti–mouse (PE; DakoCytomation); and CD27 (Alexa Fluor 700; BioLegend). Anti-CD57 (Qdot-545) and anti–PD-1 (PE) antibodies were provided by P. Chattopadhyay (Vaccine Research Center, Bethesda, MD) and G. Freeman (Dana-Farber Cancer Institute, Boston, MA), respectively. Cells were analyzed on a standard LSRII (equipped with blue, red, and violet lasers; BD Biosciences) to systematically perform eight to nine color stainings. The list mode data files were analyzed using FlowJo software (version 8.2; TreeStar, Inc.) and FACSDiva software (BD Biosciences). Multifunctional data were analyzed with PESTLE software (version 1.3.2) and SPICE software (version 3.1; obtained from M. Roederer, National Institutes of Health, Bethesda MD). Percent frequencies of multifunctional cells were calculated within the total population of detectable antigen-specific CD8+ T cells. […]

Pipeline specifications

Software tools FlowJo, SPICE
Application Flow cytometry
Organisms Human immunodeficiency virus 1, Homo sapiens
Diseases HIV Infections