Activating human T cells - (Jan/27/2006 )

Hello,

I've been trying to activate human T cells by adding PHA to PBMCs isolated by ficoll
for 2 days, and then switching to a medium that contains IL-2

I've noticed that after the PHA activation there are many attached cell aggragates (containing mostly T cells acording to FACS analysis)
My question is if this is a normal part of the procedure and when I wash the cells and switch to the IL-2 medium do I take only the suspended cells and transfer them to a new flask or do I leave them with the aggregated cells?

Thanks in advance,

Ilana

Hi-

So you see many T cell aggregates huh? They may be "blasting," a term used to describe activated T cells. They upregulate adhesion molecules after they are activated and sort of huddle together like football players

I'm not really sure what you are really asking in your post so you might want to try rewording it. Are the T cells adhering to your culture flask? What exactly are you trying to do? what are you going to do with your il-2 activated cells and which cell types are you interested in?

Hi,

Thanks for the reply,

My goal is to get a population of activated T cells (in order to further analyse certain protein expression in these cells).

After PHA activation, I see some cells that are in suspension and some in aggregates that are adhering to the culture flask. What I don't understand is if these "adhered" cells are dead /suffering cells that I should discard by transfering only the suspended cells to a new flask or if this is a normal phenotype of the activated cells. (and if so, in all further processes such as counting and washing the cells do I have to use trypsin to detach these cells?)

And while we're on the subject, what test would you suggest in order to verify that my cells are really activated?

thanks,

Ilana

Hello-


To be honest I don't usually work with PBMC's so I'm not sure whether you should take the adherent fractions or not. Given that T cells upregulate adhesion molecules on activation, some of them may be adhering to the flask. When I activate Jurkats (a human T cell lymphoma line), they usually just adhere to each other upon activation not usually the dish though. It is possible you may have other cells in your ficoll prep. If you want higher purity you could try sorting for a T cell marker or activating with an activating CD3 antibody (like 145-2c11 or OKT3).

a good activation marker is CD69 and many companies cell flow antibodies for it.

The "suffering" phenotype you observe may be, depending on your timepoint, some of your T cells undergoing activation induced cell death.

Good Luck,

Mountainman

Hi, I was activating mixed populations of T-cells (CD8/CD4 RA/RO) with PHA-P and IL-2 checked activation with TNF-alpha PCR

I also saw blasts, they sank to bottom of flask but did not adhere, are you sure they are adhering??? I usually tried to use suspension culture flasks (not treated for adherent tissue culture) but I never saw my primary human T-cells or Jurkat cells adhere after activation, even in TC treated flasks/plates -- always saw blasts that sank to bottom though... don't know why they would adhere to the flask...

i agree with mountainman too, how pure are your T-cell fractions, you indicate the attached blasts are T-cells by FACS how did you determine this, is it a subpopulation requiring trypsinization, if so then what marker did you use to determine they were T-cells after trypsinization? you would have had to permeablize and look at something internal right?? or let them recover? if you let them recover -- how long and in what media?

I just cant imagine that the T-cells were adhering to the dish.... what type of flasks do you use? are they TC treated?

Hi everybody,

Apperently I have been using TC treated flasks, which brought up the next silly question - when you grow the t cells do you place the flasks horizontally or vertically in the incubator? (as in upright or laying down?)

As for using CD69 as an activation marker, isn't cd69 considered an early activation marker that I probably wouldn't see after a week of PHA and IL-2?

I usually lay them down (ie: have larger surface area) using 5-10mL media for a T-25 flask...

What about the purity of the population are your sure the "attached" cells are T-cells and how did you confirm?

I think that in mixed PBMC there will be macrophages which willl attach, in fact the way to seperate T-cells from macrophages is to plate on TC treated and let the macrophages attach tnen take the suspension cells (should be T-cells and B-cells and maybe neutrophils and eosinophils too?) I got purified T-cells from a core facility so I don't remember exactly what all cell types are found in the first phase just after ficoll seperation... I also don't know if macrophages would blast (I would think no..) however if they do then maybe your attached blasts are macrophages...

Hope this helps you...

I am working a lot with PBMC's. I isolate the PBMC's from whole blood, using ficoll. When you want to activated your T-cells you have basicly two options:

1) Using magnetic cell sorting to sort out your T-cells (CD3+ cells) using a negative depletion. You will get a purity around 98-99%.
2) Incubate your PMCS in a flask for 2 hours or so to adhere the monocytes/macrophages. You can then collect the non-adherent cells, which are mainly T-cells. Purity of this population T-cells is much lower compaired to the sorting.

Personally I use method 2. In my experiments I use the adherent monocytes to culture macrophages and dendritic cells. And I use the suspension cells (T-cells) to activate them using anti-CD3/CD28. Activation is normally performed for 48 hours. After 24 hours you can see the cells attach to eachother which is even increased after 48 hours. In this test I do NOT see any adherent cells. the activation is measured using anti CD25-Fitc on the flowcytometer.

I performed some tests using the total PBMC fraction. Then you see clustered suspension cells but ALSO adherent cells, which are monocytes differentiated in macrophages.

When you want to do research to T-cell activation i would suggest to isolate the T-cells. In this way you prevent the possible interaction with the monocytes/macrophages in your total pbmc fraction.