Mixed Lymphocyte Reaction (MLR)

Immune checkpoints are regulatory signals that can act as ‘on and off-switches’ for T cells. Checkpoints are engaged when a T cell binds to an antigen presenting cell (APC), such as a tumor cell or dendritic cell (DC). Immunotherapies, termed checkpoint inhibitors, have been developed to target this synapse between T cells and APCs, specifically to block the immune checkpoints that usually ‘switch off’ T cells. This leads to enhanced T cell activation and increased anti-tumor response. Checkpoint inhibitor therapies currently approved for cancer treatment include anti-PD-1, anti-PD-L1 and anti-CTLA4 monoclonal antibodies, with more in active development.

MLR is an in vitro assay in which immune cells from two individuals are co-cultured together to trigger the ‘non-self’ recognition required for immune checkpoint regulation. The assay can be used to investigate the potential therapeutics by evaluating their ability to potentiate T cell response. Traditional techniques for measuring T cell response in MLR often:

  • Require a separate assay for cytokines (e.g. ELISA), proliferation and marker expression (e.g. traditional flow cytometry)
  • Use instrumentation with low-throughput acquisition that necessitate large sample volumes
  • Necessitate manual analysis of data which is compiled from multiple sources e.g. plate reader and ELISA
  • Are laborious and time-consuming, requiring steps such as protocol optimization, fixation and repetitive washes

Here we demonstrate a simple, robust workflow for measuring T cell response in MLR using the iQue® Advanced High-throughput Flow Cytometer and associated suite of validated reagents. The user can choose to combine one or more iQue® kits, including the iQue® Human T Cell Activation Kit and iQue Qbeads® Human Inflammation Panel Kit, to generate a range of outputs as desired. Enhance your screening and profiling studies by combining the power of the iQue® and the automated, integrated analysis for instantaneous pharmacological readouts for the effects of novel immunotherapies on T cell response


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Application

Assay Concept

Figure 1. Illustration of the mixed lymphocyte reaction (MLR) assay principles

In one-way MLR, isolated CD4+ T cells from one donor are combined with DCs from another donor, resulting in uni-directional T cell activation. Two way MLR is a co-culture of PBMCs from two different donors, resulting in bi-directional activation of T cells from both donors. In both assay set ups, T cell activation is dependent on an allogenic reaction with the MHC molecules on the DCs recognized as ‘non-self’. This  relies on differences in the donors’ HLA haplotypes. The induction of T cell activation response by allogenic MHCs can be enhanced with addition of checkpoint inhibitor drugs, e.g. anti-PD-1 and anti-CTLA4 monoclonal antibodies. Analysis of T cell response from one or two-way MLR can be performed using the iQue® 3 and a range of associated kits, including the iQue® Human T Cell Activation Kit, the iQue Qbeads® Human Inflammation Panel Kit and a Custom iQue Qbeads® PlexScreen Kit. These kits can be used alone or in combination to measure proliferation, marker expression and cytokine release as required to suit user needs, all from a single assay plate.

Key Advantages

Quantify surface marker & cytokine expression simultaneously

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.

Obtain quantitative data

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.

Perform high-throughput analysis

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)

Maximize your productivity

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.

MLR Assay Workflow

Figure 6. Protocol for one-way and two-way MLR assay on iQue® 3

Choose from a range of cell and bead based kits to define analysis parameters based on user needs. This assay was validated using the iQue® Human T Cell Activation Kit, the iQue Qbeads® Human Inflammation Panel Kit and a Custom iQue Qbeads® PlexScreen Kit.

Ordering Information

iQue® Human T Cell Activation Kit

Platform: Compatible with iQue® 3/ iQue® Screener Plus - VBR Configuration

Available Sizes

Catalog Numbers

1 x 96 well

90560

5 x 96 wells

90561

1 x 384 wells

90562

5 x 384 wells

90563

iQue Qbeads® Human Inflammation Panel Kit

Platform: Compatible with iQue® 3/ iQue® Screener Plus – BR and VBR Configurations

Available Sizes

Catalog Numbers

1 x 384 wells

97097

5 x 384 wells

97098

iQue Qbeads® PlexScreen: Human Secreted Proteins

Cytokine

Chemokine

TNF

Interleukin

CCL2 (MCP-1)TNF (TNFα)

IL-1α

IL-10

CCL3 (MIP-1α)sCD154 (sCD40 Ligand)

IL-1β

IL-11

CCL5 (RANTES)CD178 (Fas Ligand)

IL-2

IL-12/IL-23(p40)

CCL11 (Eotaxin)TNFβ (LT-α/TNFSF1)

IL-3

IL-12(p70)

CD14

IL-4

IL-13

CXCL9 (MIG)

IL-5

IL-17A

CXCL10 (IP-10)

IL-6

IL-17F

CXCL11 (I-TAC)

IL-7

IL-21

CX3CL1 (Fractalkine)

IL-8

IFN-α

IL-9

IFN-γ

Receptor

Adhesion Molecule

Growth Factor

Enzyme

CD121a (IL-1 RI)

CD54 (ICAM-1)

Angiogenin

Granzyme A

CD121b (IL-1 RII)

CD62E (E-Selectin)

Basic FGF

Granzyme B

TNFRI

CD62L (L-Selectin)

CSF2 (GM-CSF)

TNFRII

CD62P (P-Selectin)

CSF3 (G-CSF)

CD106 (VCAM-1)

VEGF


Resources

Product Guide

T Cell Activation Cell and Cytokine Profiling Kit

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Product Guide

Human QBeads Inflammation Panel Kit

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Application Note

Utilizing Mixed Lymphocyte Reaction (MLR) to Evaluate Checkpoint Inhibitor Therapies

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Poster

High Throughput Combinatorial Profiling of Checkpoint Inhibitor Antibodies on the iQue® Screener PLUS

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