Mixed Lymphocyte Reaction (MLR)

Multiplexed immunophenotyping and effector cytokine concentration read from a single well

Figure 2.  Measure checkpoint inhibitor drug induced cytokine release and marker expression in a single well

DCs were thawed and activated overnight (with IL-4, granzyme B and LPS) prior to plating at 40K/well in a 96 well plate. CD4+ T cells labeled with B/Green Encoder Dye were added at a 3:1 T cell-to-DC  ratio. A range of concentrations of checkpoint inhibitor drug Pembrolizumab (an anti-PD-1 monoclonal antibody) were added to enhance T cell activation. Cytokine samples were analyzed on day 2 and 6.  Marker expression was analyzed at assay endpoint (day 6). Samples were analyzed for (A) IFNγ  release, (B) TNFa release and (C) CD25 expression using the iQue^® Human T Cell Activation Kit and the iQue^® 3 platform.

Generate measurements of T cell response suitable for pharmacological analysis

Figure 3. Quantify drug induced T cell response with automatic EC₅₀ calculation by iQue Forecyt^®

DCs were seeded (40K/well) with encoded CD4+ T cells (3:1 T cell-to-DC  ratio) and treated with Pembrolizumab. 10 uL samples were analysed on Days 2 and 6 using the iQue Qbeads^® Human Inflammation Panel Kit.  (A) Heat map showing IL-2 (pg/mL) release per well. Control wells contained T cells and DCs in the absence of drug. (B-E) Curves highlight temporal and Pembrolizumab concentration dependent release of inflammatory cytokine. On Day 2, EC₅₀ for IL-2 release in response to Pembrolizumab was 0.53 ug/mL, whilst by Day 6 all IL-2 production has ceased. IL-6, CCL2 and CCL3 levels all increased from day 2 to 6, with day 6 EC₅₀ values for CCL2 and CCL3 release very similar at 0.98 and 1.0 ug/mL, respectively.

Facilitate screening and profiling studies with rapid quantification of markers and cytokines in 96 or 384-well plates.

Figure 4. Rapidly profile checkpoint inhibitor induced effects on cytokine release across multiple PBMC donor pairs using a 384 well assay format 

PBMCs (80K/well) from multiple donors were seeded alone (single donor controls) or in combination (8 donor pairs in total). Cells were treated with several concentrations of an anti-CTLA4 checkpoint inhibitor antibody. Mixed PBMCs with PHA and MMC were included as positive and negative controls, respectively. After 3 days, samples were analyzed for cytokine concentrations using IFNγ iQue Qbeads^® and concentrations calculated using a standard curve. (A) Heat map highlights differences in anti-CTLA4 induced IFNγ release between PBMC donor pairs. (B) Bar chart shows mixed PBMCs consistently released more IFNγ than single donor controls. This was enhanced with addition of 17 ng/mL anti-CTLA4. (C) Representative concentration response curve showing donor pair 7 response to anti-CTLA4 (EC₅₀ = 6.5 ng/mL)

Reduce your time to answer with real time data analysis and novel visualization tools

Figure 5.  Pre-set gating and automated data analysis by iQue Forecyt^® provides instantaneous readouts for T cell activation 

CD4+ T cells from two different donors were co-cultured separately with DCs from a single donor at a range of T:DC ratios. Each T cell donor had a different degree of mismatch with the DC donor’s HLA alleles. Donor 1 T cells were more similar in HLA profile to the DC donor with only 3/8 mismatched alleles (2 x HLA-A, -B, -C and -DR alleles) compared to the donor 2 T cells which had 7/8 mismatched alleles. Controls included T cells cultured alone (negative) or with Dynabeads or Immunocult (positive). Analysis was performed using the iQue^® Human T Cell Activation Kit. (A) Plate view shows well by well differences in CD25 expression within the CD4+ population. T cell activation increases with decreasing T:DC ratio and is greater on donor 2 T cells. This is expected as the high mismatch in HLA alleles is promoting recognition of ‘non-self’ cells. (B) and (C) Contour plots show % CD25 positive T cells from donor 1 and donor 2 (3:1 T:DC ratio). (D) Bar chart quantifying % CD25 cells in the CD4+ population.