Deep phenotyping of immune cell populations by optimized and standardized flow cytometry analyses

Abstract

Multicolor flow cytometry is a technology of choice for phenotyping of immune cells, and it can be used routinely for the follow up of patients in clinical trials. But it is challenging to define combinations of conjugated antibodies that efficiently allow the detailed analysis of major immune cell subsets and the identification of rare cell populations. In a collaborative work among the Immunology, Immunopathology, Immunotherapy (I ) laboratory, and the laboratory of immunomonitoring in oncology (L.I.O), we developed and validated 12 different 10-color flow cytometry panels that allow the deep immunophenotyping of cells from whole blood for the follow up of autoimmune and cancer patients. Here, we describe these optimized flow cytometry panels, showing that they provide the advanced analysis of T cells (including regulatory T cells), B cells, NK cells, MAIT cells, myeloid cells, monocytes, and dendritic cells. Most of the panels have been dried to improve standardization of the labeling and the entire procedure can be performed on less than 2 ml of whole blood. These deep immunophenotyping flow cytometry panels constitute a powerful tool for the monitoring of immune blood cells and will hopefully lead to the discovery of new biomarkers and potential therapeutic targets in autoimmune and cancer clinical trials. © 2018 International Society for Advancement of Cytometry.

Standardization of sampling and sample preparation for analysis of human monocyte subsets in peripheral blood

“…CD14 are all markers expressed in abundance and were used for the selection of monocytes in our gating strategy. However, in support of our targets and selected gating strategy, one recent publication presents optimised flow cytometry panels in which the monocyte panel does, indeed, contain anti -CD45, anti -HLA-DR, and anti -CD11 as a prerequisite for further monocyte gating (CD14 + and CD16 -/+ ) [423]. However, a more simple variant with HLA-DR + as the only prerequisite was published recently [172].…”

Immunological and clinical effects of low-dose interleukin-2 across 11 autoimmune diseases in a single, open clinical trial

“…Because unwanted stimulation of Teff could be deleterious in the treatment of AIDs, the demonstration of a specific effect of ld-IL2 on Tregs is of utmost importance for the treatment of AIDs caused by Teffs. Although classic immunophenotyping did not show any expansion of non-Treg T cells, we further assessed that the effects of IL-2 are Treg-specific in a group of nine patients in whom an extensive immunophenotyping was performed26 and using unsupervised analyses. Using t-distributed stochastic neighbor embedding (t-SNE) and flowMeans R packages,27–29 clusters were defined automatically based on Foxp3 and CD127 expression (figure 3A).…”

Systemic short chain fatty acids limit antitumor effect of CTLA-4 blockade in hosts with cancer

“…Unsupervised cell population analysis was performed using t-distributed stochastic neighbor embedding (t-SNE) algorithm using the online R software (version 3.5.0, cytofkit package). The "T cell 1" flow cytometry panel (CD95/CCR7/HLA-DR/ CD25/ICOS/ CD45RA/CD3/CD127/CD4/CD8); as previously described 53 was used, after setting the compensation matrix cells were manually gated using Kaluza Analysis (version 2.0) on size/granularity discrimination and then on the expression of CD3 and logicle transformation was applied to extracted data for all samples. t-SNE analyses was achieved on 8500 cells CD3 + for each sample, using T cell 1 panel markers for T cell differentiation status (CCR7/CD45RA/CD4/CD8).…”