OMIP-101: 27-color flow cytometry panel for immunophenotyping of major leukocyte populations in fixed whole blood

Abstract

This 27-color flow cytometry antibody panel allows broad immune-profiling of major leukocyte subsets in human whole blood (WB). It includes lineage markers to identify myeloid and lymphoid cell populations including granulocytes, monocytes, myeloid dendritic cells (mDCs), natural killer (NK) cells, NKT-like cells, B cells, conventional CD4 and CD8 T cells, γδ T cells, mucosa-associated invariant T (MAIT) cells and innate lymphoid cells (ILC). To further characterize each of these populations, markers defining stages of cell differentiation (CCR7, CD27, CD45RA, CD127, CD57), cytotoxic potential (perforin, granzyme B) and cell activation/proliferation (HLA-DR, CD38, Ki-67) were included. This panel was developed for quantifying absolute counts and phenotyping major leukocyte populations in cryopreserved, fixed WB collected from participants enrolled in large multi-site tuberculosis (TB) vaccine clinical trials. This antibody panel can be applied to profile major leukocyte subsets in other sample types such as fresh WB or peripheral blood mononuclear cells (PBMCs) with only minor additional optimization.

Keywords: absolute counts; clinical trials; fixed whole blood; high-dimensional flow cytometry; immune cell subsets; immunophenotyping.

Figure 1.. Hierarchical gating strategy

Representative example of manual gating strategy for fixed whole blood using BD FACSymphony A5 cytometer B. Labels in bold above graphs are used to indicate cell lineages / reference beads. Time gates were applied to the detector measuring the longer wavelength off each of the lasers, to exclude any inconsistent fluorescence patterns during sample acquisition. After exclusion of doublets, aggregates and reference beads, leukocytes were firstly selected based on SSC/FSC overlaid with CD45 and refined using CD45 and SSC parameters. Antibody aggregates were identified as extremes by plotting CD14 vs CD33 (the fluorochrome combination that most frequently allowed visualization of aggregates) and excluded. Following exclusion of CD66+ granulocytes and additional cleaning, CD66− myeloid and lymphoid cell populations were delineated based on CD33 expression. From the CD33+ myeloid gate, CD19+, CD3+ and CD56+ cells were excluded and HLA-DR+ cells were selected. Monocytes were stratified based on CD16/CD14 markers into classical (CD14+CD16−), intermediate (CD14+CD16+/low) and non-classical (CD14-CD16+) monocytes. From the double negative population (CD14-CD16−), mDCs were identified as HLA-DR+CD11c+. Expression of CD32 and CD38 was assessed on all CD33+ myeloid cells. From the lymphocyte (CD33− and SSC low) gate, B cells were defined as CD3−, CD4−, CD56− and CD19+ cells. Plasmablasts (CD38 and CD27 high) were identified among CD19+ B cells. B cells were further classified into naïve (IgD+CD27−) and memory (IgD-CD27+) populations. NK cells were identified after exclusion of CD3, CD4 and CD19 based on CD56 (dim and bright) and CD16 (positive or negative) expression. Overlay of perforin expression was visualized to confirm that CD56dim CD16+ cells were NK cells (not shown). Their cytotoxic and memory patterns were evaluated using perforin/granzyme B and CD57/CD27, respectively. CD3−, CD4−, CD19−, CD16−, CD56− lymphocytes expressing CD127 were categorized as ILCs. From the lymphocyte (CD33− and SSC low) gate, T cells were gated based on CD3 expression followed by exclusion of CD19+ B cells. γδ TCR+ T cells were further classified based on Vδ2 expression. Inclusion of perforin and granzyme B allowed evaluation of their cytotoxic potential. From the γδ TCR− T cell gate, phenotypic MAIT cells were identified based on TRAV1.2 and CD161 co-expression. Following exclusion of this lineage, NKT-like cells (CD56+) and conventional T cells (CD56−) were selected. Cytotoxic potential of CD3+CD56+ NKT-like cells was further characterized by perforin and granzyme B expression. Conventional T cells were then classified into the following populations: single positive CD4 T cells (CD4+CD8−), single positive CD8 T cells (CD4−CD8+) and double positives (CD4+CD8+). CCR7 and CD45RA were further used to stratify CD8 and CD4 T cells into naïve (CCR7+CD45RA+), central memory (CCR7+CD45RA−), effector memory (CCR7-CD45RA−) and terminally differentiated effector memory (CCR7-CD45RA+) cells. CD57, CD127 (not shown) and CD27 were also used to refine differentiation/memory subsets. Expression of proliferation (Ki67), cytotoxic (perforin, granzyme B) and activation (HLA-DR and CD38) markers was also assessed on CD4 and CD8 conventional T cells.