TITLE: MoDC immunogenicity assay
AUTHORS: Annabel J Curle, Sarah Howlett, Joanne Jones

[DESCRIPTION]
The monocyte-derived dendritic cell (moDC) immunogenicity assay can be utilised to test immunogenicity of different cell types. Similar to the mixed lymphocyte reaction (MLR) assay, cells of interest are co-cultured with T cells. Professional antigen presenting cells (APCs; moDCs) are added here to ensure optimal T cell activation in the case of immunogenic cells and improve limited sensitivity of the MLR-like assays. 

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. 

[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) 

[STEPS]
SECTION: Cell preparations and moDC differentiation - Day 0-7
1. Make PBMCs using preferred method from whole blood
SECTION: Cell preparations and moDC differentiation - Day 0-7
2. Take whole PBMCs and enrich for CD14+ cells using magnetic activated cell sorting (MACS) with CD14+ microbeads
SECTION: Cell preparations and moDC differentiation - Day 0-7
3. 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 
SECTION: Cell preparations and moDC differentiation - Day 0-7
3.1. 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 
SECTION: Cell preparations and moDC differentiation - Day 0-7
3.2. 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+ 
SECTION: Cell preparations and moDC differentiation - Day 0-7
4. Using the CD14- population, enrich for T cells using MACS with a PanT negative selection kit 
SECTION: Cell preparations and moDC differentiation - Day 0-7
4.1. If specific T cell population is required, perform MACS or FACS (fluorescence activated cell sorting) for required population
SECTION: Cell preparations and moDC differentiation - Day 0-7
4.2. Optional: collect PanT to check purity of MACS enrichment by flow cytometry 
SECTION: Cell preparations and moDC differentiation - Day 0-7
5. Stain T cells with proliferation dye as per preferred protocol
SECTION: Cell preparations and moDC differentiation - Day 0-7
6. Count then freeze the stained-T cells in FBS + 10% DMSO for the duration of the monocyte-DC differentiation 
SECTION: Cell preparations and moDC differentiation - Day 0-7
7. Prepare your "cells of interest" (COI) ready to have enough for 150,000 cells per well on day 7 
SECTION: Seeding the immunogenicity assay - Day 7 (assay Day 0)
8. Thaw proliferation dyed-T cells as per preferred protocol 
SECTION: Seeding the immunogenicity assay - Day 7 (assay Day 0)
9. Remove all media from moDCs and wash once with PBS (moDCs are adherent but aspirate gently to avoid disturbing cells) 
SECTION: Seeding the immunogenicity assay - Day 7 (assay Day 0)
10. 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 
SECTION: Seeding the immunogenicity assay - Day 7 (assay Day 0)
10.1. Note: optimisation will be required to ensure both cell types survive and proliferate as expected in the 1:1 media mix 
SECTION: Seeding the immunogenicity assay - Day 7 (assay Day 0)
11. 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 
SECTION: Immunogenicity assay - Day 7-12 (assay Day 0-5)
12. 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 
SECTION: Readouts - Day 12 (assay Day 5)
13. For cytokine assays: 
Collect 150 ul supernatant per well into a v-bottom 96 well plate
SECTION: Readouts - Day 12 (assay Day 5)
13.1. Centrifuge supernatants plate 1500 g x 10 minutes then re-collect supernatants avoiding any pellet/debris that have collected 
SECTION: Readouts - Day 12 (assay Day 5)
13.2. Freeze supernatants at -80oC until use. 

Perform ELISA or luminex cytokine assays including IFNg and TNFa as readouts of immunogenicity. 
SECTION: Readouts - Day 12 (assay Day 5)
14. 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). 
SECTION: Readouts - Day 12 (assay Day 5)
14.1. 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