P2X7 study Immunophenotyping assays

1. P2X7 study Immunophenotyping assays

2. Screening Dry ice Italy Dry ice Cambridge £10 PAXgene DNA £10 PAXgene RNA £10 PAXgene RNA DNA for WGS SNP genotyping P2X7/IL1b/GR gene expression £30 £100 ‘Spare’ RNA for whole blood transcripts IN/OUT Cost: £160

3. Visit EDTA 20-60ml EDTA tubes purple top Serum 2.5ml SST Plasma 2.5ml SST Pre-filled LPS tube 1 ml? Dilute, aliquot, stimulate (24hrs?) Spike in ATP 4 hours Layer and isolate buffy coat Spin and store at -80 PBMC LPS/bzATP TruCount LPS/DEX stimulation MACS Spin and store at -80 £20-30 Cytokines £300 £10 Sorted cells £100 Spin and store at -80 IL1b £25 Store in RLTplus in -80 Whole blood cytokines (cytokine secretion potential) Cryopreserve Store at -80 Transcripts £50-300 Blood kit £250 each cell type. 4 cell types £30 Immunophenotype (deep?) Monocyte classes expressing P2X7 and GR QC FACS £30 £200 The following day everything is shipped to Cambridge on dry ice Upfront costs: ~£410 per participant with ~£200 for phenotyping committed before starting Note: MACS price assumes 3 separations

4. What would this look like at the site lab? DAY 1 • Bloods taken in morning and delivered to lab within half an hour • bzATP are spiked into the special tube • 37c incubator 4 hours • Tubes spun down and supernatant collected and stored at -80 • Whole blood diluted and aliquoted into wells with simulation cocktails. Incubated 24 hours • SST and PPT tubes spun down and serum/plasma collected and stored at -80 • Remaining purple top EDTA tubes used for PBMC isolation, as per BIODEP protocol • Blood is layered and buffy coat collected • Aliquot taken and stored in RLTplus (for PBMC transcripts) at -80 • Aliquot taken and subsets purified by MACS, stored in RLTplus -80 • Remaining aliquots cryopreserved and stored at -80 • QC FACS performed on subsets • TruCount performed simultaneously DAY 2 • LPS/DEX plates spun and supernatant put into dry ice package with everything else to be shipped to Cambridge • Requires a dedicated lab person and 1.25 full days • Very similar to the BIODEP protocol already in place • All lab requirements for BIODEP apply

5. And at the Cambridge lab • Serum, plasma, supernatants from LPS and ATP stimulations, RLTplus all stored at -80 for batch analysis at a later date • These can then either be analysed in Cambridge or shipped off to their final homes in one batch at the end. • Cryopreserved PBMCs stored in Liquid nitrogen until all sample for that participant have been collected (8 weeks) • All samples from participant thawed and analysed on the same day, with a control • Deep immunophenotyping? • PrimeFlow analysis of monocyte subsets and P2X7, GR (room for one more, maybe IL1b) mRNA expression

6. Visit – Without purifying cell subsets Pre-filled Ficoll tubes 10-20 ml EDTA 5ml Serum 2.5ml SST Plasma 2.5ml SST Pre-filled LPS tube 1 ml? FACS Spike in ATP 4 hours Dilute, aliquot, stimulate (24hrs?) Spin and tip Spin and store at -80 TruCount LPS/bzATP £20-30 LPS/DEX stimulation Spin and store at -80 Cytokines £10 Buffy Coat £100 Spin and store at -80 IL1b £25 Store in RLTplus in -80 Whole blood cytokines (cytokine secretion potential) Cryopreserve Store at -80 £50-300 Blood kit £30? Transcripts Immunophenotype (deep?) Monocyte classes expressing P2X7 and GR £250 £200 The following day everything is shipped to Cambridge on dry ice Upfront costs: ~£90 per participant with ~£200 for phenotyping committed before starting

7. What would this look like at the site lab? DAY 1 • Bloods taken in morning and delivered to lab within half an hour • bzATP are spiked into the special tube • 37c incubator 4 hours • Tubes spun down and supernatant collected and stored at -80 • Whole blood diluted and aliquoted into wells with simulation cocktails for LPS/DEX assays. Incubated 24 hours • Perform TruCount • SST and PPT tubes spun down and serum/plasma collected and stored at -80 • Ficoll tubes spun and buffy coat collected • Take small aliquot for storage in RLTplus at -80 • Cryopreserve remainder DAY 2 • LPS/DEX plates spun and supernatant put into dry ice package with everything else to be shipped to Cambridge • Requires a lab person and a half day, with ~1 hour to spin and prepare package on day 2. Much less involved and there is less room for error. Not as much training required • Requires Cat2 hood, 37c incubator, centrifuge with plate rotor, -80 freezer and FACS machines

8. Immunophenotyping • Fluorescence based immunophenotyping • Use FACSsymphony (24 colours available) • ADAPTIVE: T cells, B cells, Tregs, specific markers of interest • INNATE: Monocytes, NK cells, DCs, NKT, ILC, gdT cells specific markers of interest (expanded panel to capture far more innate immune cell populations than before) • CCR2,GR, P2X7: Specific markers of interest that we can measure expression levels of (not just numbers of cells) • 2 newly designed panels would give us relative proportions of all the major subsets of cells, and we could compare expression levels of 3 markers across cell types, samples and across time points • Does the expression of the GR or P2X7 differ in different cell types? How do the expression levels of GR and P2X7 correlate in different patients? Do they change after treatment? • Is there something in the immunephenotype that predicts treatment response?

9. PrimeFlow • Combined flow cytometry and mRNA gene expression method • Stain for surface markers (monocyte subsets) • Intracellular stain the mRNA of 3 target genes and 1 reference gene • Relative quantification of gene expression by specific cell subsets • Ie. Which cell types are responsible for the P2RX7 or GR etc. gene expression signature • Are they the ‘immature monocytes’ and how does this change before and after treatment

10. To Do • DEX and LPS assays need to be optimized and tested for feasibility in multisite (1-2 months, START BY JULY) • TruCount + monos needs to be optimized (1 month, 2 months for reagents to come in, STARTY BY JUNE) • LPS + bzATP assay needs to be tested in our hands (1 month, START BY JULY) • Immunophenotyping and PrimeFlow optimized (3 months minimum, START BY JUNE)