Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Abstract

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

Figure 1:. Titration of in-house conjugated antibody-metal conjugates for NK panel.

Titrations of in-house conjugated antibodies were performed on PBMCs from a healthy donor using five different concentrations: 0.625, 1.25, 2.5, 5, and 10 μg/mL. Titers for anti-CD3, anti-CD14, anti-CD33, anti-CD19, anti-PD-1 and anti-CD56 were determined by gating on live cells. Titers for anti-CD4 and anti-CD8 were determined by gating on T cells. Titers for the remaining antibodies were determined by gating on NK cells. Since NKp44 is not expressed on resting NK cells, titers were determined on PBMCs stimulated with IL-2 and shown on NK cells. The red arrows indicate the titer selected for each antibody.

Figure 2:. Titration of in-house conjugated ligand panel antibodies.

Titrations of in-house conjugated antibodies were performed on PBMCs from a healthy donor using five different concentrations: 0.625, 1.25, 2.5, 5, and 10 μg/mL. Titers for anti-HLA-DR, anti-ICAM-1, anti-CCR2, anti-CD14, anti-CD11b, and anti-LFA-3 were determined by gating on CD3⁻CD7⁻ cells. Titers for anti-CD3, anti-pan HLA class I, anti-CD7, anti-CD48, anti-LLT-1, anti-HLA-C,E, anti-HLA-E, anti-FasR, anti-Nectin-1, anti-MICA/MICB, anti-DR4/DR5, anti-ULBP-1,2,5,6, anti-Nectin-2, anti-CD155, anti-HLA-Bw4, anti-HLA-Bw6, anti-CD33, anti-CD56, and anti-B7-H6 were determined by gating on live cells. Titers for anti-CD4 and anti-CD8 were determined by gating on CD3⁺ cells. The red arrows indicate the titer selected for each antibody.

Figure 3:. NK panel gating strategy and performance.

(A) Serial negative gating from whole PBMCs to NK cells is shown in a healthy donor. Intact, bead and event-length gates ensure successful gating to single cells. Cisplatin staining was performed as a Live/Dead stain. T cells and B cells were excluded using CD3 and CD19. Monocytes were excluded by negative gating on CD4 and CD14/CD33 and by further negative gating of CD56-/HLA-DR^bright cells. CD56 and CD16 were used to identify different subsets of NK cells (CD56^bright, CD56^dim and CD56⁻). (B) Examples of expression of NK cell receptors on NK cells from one healthy donor purified by magnetic-bead isolation.

Figure 4:. Ligand panel gating and performance.

(A) Gating of major immune cell subsets from PBMCs derived from a healthy donor following normalization, calibration bead removal, and debarcoding. (B) Expression of ligands for NK cell receptors as well as several myeloid markers on live PBMCs. Staining for all ligands except Nectin-1 and B7-H6 is shown on PBMCs from acute dengue patients. Staining for Nectin-1 and B7-H6 is shown on PBMCs from HIV-infected individuals who were virologically suppressed.