Feb 27, 2023
Version 2
The monocyte-derived dendritic cell (MoDC) assay – an in vitro assay for testing the immunogenicity of cellular therapies V.2
- 1University of Cambridge
Protocol Citation: Annabel J Curle, Sarah Howlett, Joanne Jones 2023. The monocyte-derived dendritic cell (MoDC) assay – an in vitro assay for testing the immunogenicity of cellular therapies. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jmqdl1b/v2Version created by Annabel J Curle
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 27, 2023
Last Modified: February 27, 2023
Protocol Integer ID: 77698
Keywords: Immunogenicity, T cell, In vitro, monocyte-derived DC
Funders Acknowledgements:
MRC UKRMP
Grant ID: G101985
Abstract
The monocyte-derived dendritic cell (MoDC) assay can be used to test, in vitro, the immunogenicity of potential regenerative medicine cellular therapies. Similar to a mixed lymphocyte reaction (MLR), cells of interest (COI) are co-cultured with T cells, but with the addition of induced professional antigen presenting cells (APCs; moDCs) to ensure optimal T cell activation and to improve the limited sensitivity of the MLR-like assay.
Autologous CD14+ monocytes and T cells are isolated from PBMCs. Monocytes are differentiated to mature dendritic cells over 7 days, then co-cultured with T cells and cells of interest for 5 days. Recommended readouts of the assay are ELISA (for IFNg/TNFa release) and flow cytometry (for T cell proliferation and CD25 expression).
Positive controls wells of allogeneic moDC/T cells and bead-activated T cells are utilised. Biological replicates can be run in parallel. Well-well variation is minimal, but samples can be run in technical duplicates if desired.
Materials
Cell preparations:
PBMC isolation materials
MACS equipment and LS tubes
CD14+ microbeads for MACS (we use Miltenyi CD14+ microbeads)
PanT cell isolation kit for MACS (we use Miltenyi PanT cell isolation kit)
96 well tissue culture plates
Cell proliferation dye (we use eBioscience™ Cell Proliferation Dye eFluor™ 450)
Cell freezing media (FBS + 10% DMSO)
moDC differentiation
RPMI
Penicillin/streptomycin
Human AB serum
rhIL-4
rhGM-CSF
LPS
PBS
Co-culture
Cell of interest media
T cell activation beads (we use Gibco Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation)
Readouts
ELISA materials
Flow cytometry materials
Before start
Fresh blood must be used each time as monocytes do not survive the freeze/thaw process
Optimisation experiments must be performed to ensure all cell types survive without compromise to survival, proliferation and differentiation state in the 1:1 mix of medias (T cell/moDC media: cell of interest media)
Cell preparations and moDC differentiation - Day 0-7
Cell preparations and moDC differentiation - Day 0-7
1w 0d 3h
1w 0d 3h
Make PBMCs using preferred method from whole blood
Take whole PBMCs and enrich for CD14+ cells using magnetic activated cell sorting (MACS) with CD14+ microbeads
Count monocytes, then seed in a 96 well plate at 150,000 cells/well in 200 ul RPMI + 10% P/S + 5% human AB serum (HAB) + 50 ng/ml rhIL-4 and 50 ng/ml rhGM-CSF (day 0)
Incubate in 37oC incubator for 7 days refreshing half media on day 3 and 6
Each media refresh should be done with double concentration IL-4 and GM-CSF to account for half media change: remove 100 ul media, add 100 ul RPMI + 10% P/S + 5% HAB + 100 ng/ml rhIL-4 and 100 ng/ml rhGM-CSF
Day 6 media change, plus 100 ng/ml LPS for activation : remove 100 ul media, add 100 ul RPMI + 10% P/S + 5% HAB + 100 ng/ml rhIL-4 and 100 ng/ml rhGM-CSF + 200 ng/ml LPS
Optional: collect monocytes (day 0), immature moDCs (day 6) and mature moDCs (day 7) to check purity of MACS enrichment by flow cytometry
Monocytes: CD14+
Immature DCs: CD14-, CD80+, CD86+, HLA-ABC+, HLA-DR+
Mature DCs: CD14-, CD80+, CD86+, HLA-ABC+, HLA-DR+, CD83+
Using the CD14- population, enrich for T cells using MACS with a PanT negative selection kit
If specific T cell population is required, perform MACS or FACS (fluorescence activated cell sorting) for required population
Optional: collect PanT to check purity of MACS enrichment by flow cytometry
Stain T cells with proliferation dye as per preferred protocol
Count then freeze the stained-T cells in FBS + 10% DMSO for the duration of the monocyte-DC differentiation
Prepare your "cells of interest" (COI) ready to have enough for 150,000 cells per well on day 7
Seeding the immunogenicity assay - Day 7 (assay Day 0)
Seeding the immunogenicity assay - Day 7 (assay Day 0)
2h
2h
Thaw proliferation dyed-T cells as per preferred protocol
Remove all media from moDCs and wash once with PBS (moDCs are adherent but aspirate gently to avoid disturbing cells)
Experimental wells:
Seed 150,000 autologous T cells per well in 150 ul RPMI + 10% P/S + 5% HAB
Seed 150,000 COI per well with 150 ul media specific to cells of interest (+ ROCKi if required)
Final: 1:1:1 ratio of moDC:T cell:COI in 300 ul of 50:50 media RPMI media: COI media
Note: optimisation will be required to ensure both cell types survive and proliferate as expected in the 1:1 media mix
Positive control wells:
(1) Allogeneic Tcell-moDC
Seed 150,000 allogeneic T cells into moDC wells in 150 ul RPMI + 10% P/S + 5% HAB
Add 150 ul media specific to cells of interest
(2) Polyclonally activated T cells
Seed 150,000 T cells into an empty well (no moDC) in 150 ul RPMI + 10% P/S + 5% HAB
Add activation beads at recommended ratio in 150 ul media specific to cells of interest
Immunogenicity assay - Day 7-12 (assay Day 0-5)
Immunogenicity assay - Day 7-12 (assay Day 0-5)
5d
5d
Incubate co-culture for 5 days, refreshing half media every 48 hours (day 2 and day 4)
Each change: remove 150 ul media and add 150 ul prepared 1:1 media
Readouts - Day 12 (assay Day 5)
Readouts - Day 12 (assay Day 5)
2d
2d
For cytokine assays:
Collect 150 ul supernatant per well into a v-bottom 96 well plate
Centrifuge supernatants plate 1500 g x 10 minutes then re-collect supernatants avoiding any pellet/debris that have collected
Freeze supernatants at -80oC until use.
Perform ELISA or luminex cytokine assays including IFNg and TNFa as readouts of immunogenicity.
For flow cytometry:
Collect remaining supernatants containing non-adherent T cells by agitating gently, washing the well 1-2 times with the supernatant.
Note: moDCs are adherent so will not be collected, consider that COI may be adherent or non-adherent (it is ok if some are collected as they will be gated out during flow cytometry).
Perform flow cytometry immediately, including a minimum panel of: CD3, CD4 and CD8 (T cell markers), CD25 (activated T cell marker) and live/dead stain.
Note: cells are already pre-stained with proliferation dye
Activated T cells (CD25+, proliferation dye low/negative) are used as readout of immunogenicity of COI