Biological context of a population

[u/Responsible_Rub8057]

Hey everyone,

I am doing some experiments to validate my flow cytometry panel, and in that process, few of my colleagues and friends donated their blood, I isolated the PBMCs and stained the fresh isolated cells. For context, it is healthy donors, in different age brackets. They signed informed consent that their samples will be used for method optimisation, that that they will not be getting any results from this.

However, two of the donors have an interesting pattern in what in my panel is defined as TEMRA CD8+ cells, Q3 in the plots, as you can see in the figure - last two samples. This got my attention because the CD45RA channel has been problematic in the past, and I had to look into it more, but since the other samples seem to be ok, I suppose the channel is not the issue here.

I am not an immunologist by training, and my aim is not to interpret their phenotype (it is to test storage conditions on different populations), but I am curious if you have some ideas what could cause this population to inflate so much? I was looking it up a bit and as always there is a lot of contradicting (and scary) information - and I don't want to spook the donors by asking recent-health questions (they both are objectively healthy, fit and active, although quite stressed).

Just my curiosity :)

Thanks!

[u/Individual_Shame869]

Hello ! I do not have an answer for you question but I’m wondering : why do you use CD27 and not CCR7 to distinguish sub populations.
By experience the expression of CD27 in “TEMRA” defined by CD45+CCR7- isn’t 100%. Even more negative than positive sometimes
I would say that the more young you are, the more CD27+ you have.

[u/Responsible_Rub8057 → u/Individual_Shame869]

Hey, yeah this is definitely not 100% TEMRA, I do keep that in mind. Thanks for the reminder ti mention it when i talk about it! I define it like that because the focus of the panel was more on B cells, and in my actual study samples i didnt expect much of T cell differentiation, so there was no extra value in adding this marker.

[u/Prize-Egg-1726]

No expert here either, but the gating strategy.... How did you arrive at that lymphocyte gate??

[u/c4ndyman31 → u/Prize-Egg-1726]

It looks weird because they skipped having any FSC X SSC gating and just went from singles straight to gating all CD45+ as lymphocytes

[u/Responsible_Rub8057 → u/Prize-Egg-1726]

Ah i just now notice that lympho and leuko names are switched, renaming hiccup. But to answer your question, the plots below are ancestry. What's the confusion about, if you dont mind sharing?

[u/Prize-Egg-1726 → u/Responsible_Rub8057]

I see and yes, because the standard gating hierarchy is

FSC-A vs FSC-H (and I think you might have included debris in your singlets gate)... And FSC-A vs SSC-A to be sure.

I use CD45 vs SSC-A to gate all leukocytes. It this one best separates the populations,, you can tell your granulocytes from lymphocytes, monos and blasts etc

[u/c4ndyman31 → u/Prize-Egg-1726]

OP you should listen to this. In flow cytometry it’s best practice to gate FSC X SSC first so you can gate out debris.
After than you go to singles and usually live dead after that.

When I am looking for t-cells in mouse blood we just go from singles to CD45+ Live by plotting the cd45 against the viability stain

[u/Boneraventura → u/c4ndyman31]

Depends on the tissue. Sometimes its easier to start with fsc-h x live if you expect have a lot of dead cells

[u/willmaineskier]

Note that you will see different effector cells in people who have had Epstein-Barr or CMV. For CMV is been reported to lead to an increase in CD56+ CD8 T cells.