OMIP‐056: Evaluation of Human Conventional T Cells, Donor‐Unrestricted T Cells, and NK Cells Including Memory Phenotype by Intracellular Cytokine Staining

Abstract

A 26‐color staining panel was developed to profile human antigen‐specific T cells in an intracellular cytokine staining (ICS) assay using peptide pools to various antigens of interest. In addition to multiple functional markers, the panel includes differentiation/activation markers and markers to assess γδ, mucosal‐associated invariant T, and NK T cells as well as conventional NK cells. Panel optimization was performed using previously cryopreserved PBMC from healthy adults, and then, expression of key functional markers in the panel was cross‐validated against a validated ICS assay used in the HIV Vaccine Trials Network (HVTN). The panel is currently being used to evaluate the responses to tuberculosis and malaria vaccine candidates in volunteers from different geographic areas. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

A single mRNA immunization boosts cross-variant neutralizing antibodies elicited by SARS-CoV-2 infection

“…Flow cytometry was used to examine SARS-CoV-2-specific CD4+ T-cell responses using a validated ICS assay. The assay was similar to published reports( 84, 85 ) and the details of the staining panel are included in Supplemental Table 2. Two peptide pools (15 amino acids overlapping by 11 amino acids, provided by BioSynthesis) covering the spike protein of SARS-CoV-2 were used for the six-hour stimulation.…”

A phase 1b randomized study of the safety and immunological responses to vaccination with H4:IC31, H56:IC31, and BCG revaccination in Mycobacterium tuberculosis-uninfected adolescents in Cape Town, South Africa

“…Briefly, cryopreserved PBMC were thawed, incubated overnight and stimulated on day 2 for six hours at 37°C with either peptide pools (peptides of 15 amino acids overlapping in sequence by 11 amino acids) for the vaccine-matched proteins (Ag85B, ESAT-6, Rv2660c and TB10.4), BCG (Pasteur strain grown from glycerol stocks and provided by Aeras, Rockville, MD), dimethyl sulfoxide (DMSO, 0¢5%, Sigma Aldrich, Saint Louis, MO; negative control) or staphylococcal enterotoxin B (SEB, 0¢25 mg/mL, Sigma Aldrich; positive control) in the presence of costimulatory antibodies CD28 and CD49d (1 mg/mL, BD Biosciences, San Jose, CA) and brefeldin A (BFA, 10 mg/mL, Sigma Aldrich, Saint Louis, MO). Cells were incubated with ethylenediaminetetraacetic acid (EDTA, 2 mM, Life Technologies) overnight at 4°C, then stained with a 26-color antibody staining panel (modified version of [17]) and acquired on a BD FACSymphony A5 flow cytometer (BD Biosciences, San Jose, CA), and analyzed using FlowJo version 9¢9¢6 (BD, Franklin Lakes, NJ). Data would have been excluded from analyses if fewer than 1000 CD4+ and/or CD8+ T cells were counted; however, no samples were excluded based on this criterion.…”

Analytical performance of a 25‐marker spectral cytometry immune monitoring assay in peripheral blood

“…The advantages of spectral cytometry [5], spectral unmixing [6], and guidance on spectral cytometry panel design [7] have been reported elsewhere. We aimed for a 25‐biomarker immune monitoring panel, using OMIP‐042 [8] as a starting point while referencing major immunophenotypes described by the Human Immune Phenotyping Consortium [1], OMIP‐034 [9], and OMIP‐056 [10]. Ultimately, we reviewed over 45 OMIPs and immune panels published within the last decade.…”