Population analysis and immune cell killing

o2h offers cancer immunotherapy drug discovery research services, which range from biochemical and cell-based assays for the phenotypic screening of novel immune therapeutics and the in vitro determination of the molecular mechanism of action of new anti-cancer immune modulators.

At o2h, we have developed a repository of healthy human donor derived peripheral blood mononuclear cells (PBMCs) which serve as starting material for various downstream immuno-oncology assays. These cells have been characterized by flow cytometry based immunophenotyping for multiple immune cell subsets using our in-house, state of the art flow cytometer CytoFLEX S from Beckman Coulter, which comes equipped with 4 lasers and allows detection up to 15 channels.

• o2h scientists can utilize flow cytometry-based analysis to perform immune phenotyping of whole blood-derived PBMCs and quantify the expression of surface receptors and intracellular proteins.
• Co-culture systems, such as PBMCs, T cells, and NK cells, can be characterized by their paracrine signalling, activation, proliferation, and differentiation using fluorochrome-conjugated antibodies against cell-surface receptors.
• Therapeutic small molecules and antibodies can also be screened for their cytotoxic potential against cancer cell lines using Annexin V/7-AAD dyes to quantify the percentage of cellular apoptosis and necrosis

Figure 1: Human PBMCs stained with a T cell antibody panel. The gating strategy involved first identifying the lymphocyte population by forward scatter (FSC) and side scatter (SSC). Live T cells (CD3+) were gated and then the two main types of T cells were defined by CD4+ (T helper cells) and CD8+ (cytotoxic T cells).

Lymphocyte-mediated cytotoxicity is a form of cellular immunity targeting intracellular pathogens. Lymphocyte-mediated cytotoxicity assays are crucial to understand the potency of small molecule and antibody-based immunotherapies. The most popular in vitro methods to monitor lymphocyte-mediated cytotoxicity of target cells are cell-mediated cytotoxicity assays such as ADCC (antibody-dependent cellular cytotoxicity) and TDCC (T cell-dependent cellular cytotoxicity), in which immune effector cells and target cells are co-cultured.

The CFSE/7-AAD Cytotoxicity assay is designed to determine the cytotoxicity profile of PBMC (Peripheral Blood Mononuclear Cells) towards target cells of interest. It consists of CFSE (Carboxyfluorescein succinimidyl ester), used to identify target cells in a mixed cell population of target cells and PBMC, and 7-AAD (7-aminoactinomycin D) to label dead cells. CFSE and 7-AAD labelling enables cell cytotoxicity detection at the single-cell level using flow cytometry.

At o2h, we have employed the above cytotoxicity assay to show that small molecule PD-1/PD-L1 checkpoint inhibitor BMS-1166 is able to rescue PBMC mediated cytolysis of MDA-MB-231 breast cancer cells using a PBMC cancer cell co culture system.

Figure 2: Breast cancer cell line MDA-MB-231 was evaluated for cell surface expression of checkpoint ligand PD-L1 using flow cytometry. Cells were stained with LIVE/DEAD Fixable Violet Cell Stain followed by incubation with anti-PD-L1 antibody (1:100 dilution) for 30 minutes on ice. Cells showed (A) around 90% expression and (B) 4-fold higher PD-L1 median fluorescence intensity (MFI) compared to isotype control

Figure 3: Cytolytic activity of small molecule PD-1/PD-L1 checkpoint inhibitor in CFSE 7-AAD cytotoxicity assay. CFSE-labelled target cells MDA-MB-231 were incubated with effector human PBMCs at a 5:1 effector-to-target (E:T) cell ratio for 72 hours in the presence of IL-2 (200 IU/ml) and anti-CD28 antibody (1µg/ml) without (A) or with (B) PD-1/PD-L1 checkpoint inhibitor BMS-1166 at 10 µM concentration. Cell lysis was measured by the percentage of CFSE+/7-AAD+ MBA-MB-231 cell population. (C) BMS-1166 induced 4-fold higher lysis of target cells compared to the DMSO control, indicating rescue of PD-1/PD-L1 checkpoint blockade.

To know more about our immuno-oncology drug discovery services or to request our brochure, please reach out to us at discovery@o2h.com.