Generate data at MUSC

[hammer]

Some questions

• Will FACS sorting disturb RNA?
• How should we preserve the cells post-sorting and pre-RNA isolation?
• How should we isolate RNA?
• What markers should we sort on?

[michellehnelson]

Sorry, I meant CD8. I'm just conditioned to type CD4.

[hammer]

From @ChrystalPaulos:

One final thought: In the Ahmed 2011 paper, I noticed that the sorted PD-1+ and PD-1 negative CD8 T cells were stimulated with either CD3/CD28 beads or with PMA/IONO. This is very generic simulation and does not account for the role of PD-L1 signal. Thus I am not surprised both subsets could secrete IFNg, Granzyme B and perforin.

What I think would be a better experiment is to include PD-L1 ligation impact on these cells. James Riley at Penn has given use an artificial antigen presenting cells (K562s) that express CD3 agonist, CD28 agonist and PD-L1 - I think using this reagent (that test the power of the PD-1/PD-L1 interaction) instead of beads or PMA/Iono that bypass this axis would better simulate the suppressive nature of PD-1+ versus PD-1- CD8+ T cells. Something you could follow up with next year if you want...

[ChrystalPaulos]

It looks like both of the companies that sale antibodies for PD-1 stimulate their CD8+ T cells with PHA. Not really fair. Will check to see if Ahmed paper did this prior to sort.

[ChrystalPaulos]

This 2009 paper by Mojgan, Rosenberg and crew gives a very different impression of PD-1 on the peripheral blood, tumor and normal tissue on T cells in cancer patients. Instead of 60% PD-1 it is maybe 6% PD-1 on CD8s. Not sure what to believe....

Mojgan PD-1 in blood, tumor and normal tissue.pdf

[hammer]

We decided to sort into 3 subsets: PD-1-, PD-1+CD39+, and PD-1+CD39-. PD-1+CD39+ is quite rare; Zach estimated we'd have ~100k cells. We'll have to look into a low input kit for these cells.

[hammer]

How much RNA does a typical mammalian cell contain?: a typical mammalian cell contains 10–30 pg total RNA; mRNA accounts for only 1–5% of the total cellular RNA. So 0.1 - 1.5 pg of mRNA/cell, and if we have 100k cells, we should have 10 - 150 ng of mRNA

I've seen 300 ng given as a minimum input amount for RNA-Seq, so we'll definitely need a low input kit for the PD-1+CD39+ population.

https://www.ncbi.nlm.nih.gov/pubmed/25649271 says that excellent data can be recovered with even the 50 pg input total RNA with RNA amplification kits, so we should be good.

[hammer]

Data from this week's extraction!

ND257

Subset	Amount	A260/280 (purity)
Bulk CD8+	13.7 ng/ul	1.8
PD1-	39.3ng/ul	1.95
PD1+ CD39+	7.7 ng/ul	1.53
PD1+ CD39-	27.5 ng/ml	1.93

ND258

Subset	Amount	A260/280 (purity)
Bulk CD8+	13.1 ng/ul	1.79
PD1-	44.9 ng/ul	2.02
PD1+ CD39+	5 ng/ul	1.94
PD1+ CD39-	24.2 ng/ul	1.94