A flowcytometric analysis to efficiently quantify multiple innate immune cells and T Cell subsets in human blood

Abstract

The balance of inflammation and immunosuppression driven by changed ratios in diverse myeloid and T cell subsets, as well as their state of activation and ability to migrate to lymphoid compartments or inflammatory sites, has emerged as a highly active area of study across clinical trials of vaccines and therapies against cancer, trauma, as well as autoimmune and infectious diseases. There is a need for effective protocols which maximally use the possibilities offered by modern flow cytometers to characterize such immune cell changes in peripheral blood using small volumes of human blood. Additionally, longitudinal clinical studies often use cryopreserved samples, which can impact flow cytometric results. To efficiently gauge both the innate and the adaptive immune response, two novel 15-color antibody panels to identify key myeloid and T cell subsets and their functional potential were established. This approach was used to compare cellular immune profiles in fresh whole blood and in matched cryopreserved peripheral blood mononuclear cells (PBMCs). Cocktail I was designed to identify and characterize myeloid cell populations including dendritic cells (DCs), monocytic monocyte-derived suppressor cells (MO-MDSC), and monocytes, determining further core aspects of their state of maturity, T cell stimulatory (or inhibitory) potential, and migration capability. Cocktail II was used for phenotyping diverse T cells subsets, and their key migration and functional regulatory capabilities. The two 15-color antibody panels for the evaluation of both immune-stimulating and immunosuppressive processes presented herein allowed for efficient evaluation of the balance of immune activation versus immunosuppression across key blood cells, with good resolution for all 15 markers stained for in each panel. Gating strategies for the myeloid and T cells are presented to further support specific subset identification. This protocol was shown to be reproducible across donors and useful to study both RBC-lysed whole blood and cryopreserved PBMCs. © 2017 International Society for Advancement of Cytometry.

The early in‐vivo effects of a single anti‐emetic dose of dexamethasone on innate immune cell gene expression and activation in healthy volunteers

“…After thawing, peripheral blood mononuclear cell were washed twice and transferred to a 96-well roundbottom plate (Corning, Corning, NY, USA) to be stained with a fluorochrome label. We have recently established two antibody cocktails for surface staining of peripheral blood mononuclear cells to assess the cellular innate immune response [9]. The assessed myeloid and lymphoid cell populations as well as functional markers were chosen as they represent key cells/markers involved in inflammation and immune stimulation as well as immunosuppression (see also Supporting Information, Table S2).…”

Effect of delayed cell processing and cryopreservation on immunophenotyping in multicenter population studies

“…Several previous studies have shown no effect of cryopreservation in the enumeration of T cells, helper CD4+ and cytotoxic CD8+ T cells 2, 7, 8, 11, 12 . These results are largely consistent with the results of our study where we did not find any pairwise differences in percentage of total T cells unless cells were cryopreserved after a delay in cell processing for 72 hours.…”

Conventional and Computational Flow Cytometry Analyses Reveal Sustained Human Intrathymic T Cell Development From Birth Until Puberty

“…Using manually defined gates, we examined the CD45 + fraction of human thymic MNC in order to identify populations of CD19 + HLA-DR + B cells, HLA-DR + CD123 + plasmacytoid dendritic cells (pDCs) and HLA-DR + CX3CR1 + conventional dendritic cell and macrophages (cDC/macro) ( Figure 2A ) and HLA-DR + CD14 + monocytes ( Figure S1A ) ( 23 , 24 ). These populations were used to assess the performance of our graph-based clustering approach, which appeared to successfully identify the distinct populations ( Figure 2B and Figure S1B ) and which recapitulated the phenotype of manually identified mature non T-lineage subsets based on both the biexponentially transformed scatterplots ( Figure 2C and Figure S1C ) and the median fluorescent indices (MFIs) of each of the annotated clusters ( Figure 2D ).…”