hi

I think we need a lot more detail about your set-up to know how to answer you.

first, what antigen are you using? is it one that humans are routinely immunized to? if not, then the frequency of responding cells in your primary cultures will be very low. you'll need at least to add some IL-2 to the cultures to keep them alive.

second, if you re-stimulate them at day 2, you are likely to get something called "antigen induced cell death".

third, when you "rest" them, does that mean that you have washed the antigen away? or is it still there? if you washed the cells and didn't add IL-2, many (most) of them will die, including any that have responded to your original antigen.

fourth, is your human serum from blood type AB donors? if not, you could have some problems with antibodies. did you heat-inactivate it to remove complement activity?

it might be good for you to carefully read the materials and methods in a whole bunch of papers to see if your system matches up to those in which people area successful at growing out antigen-specific T cells. you could start with some of the early papers by Antonio Lanzavecchia.

cheers. polly

hi

I think we need a lot more detail about your set-up to know how to answer you.

first, what antigen are you using? is it one that humans are routinely immunized to? if not, then the frequency of responding cells in your primary cultures will be very low. you'll need at least to add some IL-2 to the cultures to keep them alive.

second, if you re-stimulate them at day 2, you are likely to get something called "antigen induced cell death".

third, when you "rest" them, does that mean that you have washed the antigen away? or is it still there? if you washed the cells and didn't add IL-2, many (most) of them will die, including any that have responded to your original antigen.

fourth, is your human serum from blood type AB donors? if not, you could have some problems with antibodies. did you heat-inactivate it to remove complement activity?

it might be good for you to carefully read the materials and methods in a whole bunch of papers to see if your system matches up to those in which people area successful at growing out antigen-specific T cells. you could start with some of the early papers by Antonio Lanzavecchia.

cheers. polly

Hi,

I presume that you do not stimulate your control sample. If so, how do you keep T cells in that same control sample alive for 5-9 days to compare them with T cells in your treated sample? Are you using any cytokines in your control sample to keep them alive? In that case you should control your experiment accordingly.

Cheers

This answer might be simple, but is in my view very relevant:

What is or how do you define memory?

Can you recapitulate T cell memory at all in vitro?

Do you need antigen in vitro and/or in vivo?

For the cultures you may add IL-2, IL-7, IL-15 or stromal cells!

In addition to the above comments, I would suggest you look into how you are detecting antigen specific proliferation of T cells in treated vs control?

Also, if you are looking into cytokine production of T cells; how did you control for cytokine production in the other cells...irradiation ? mitomycin c? I use murine immune cells and have noticed that Monocytic cells can produce enough cytokine to cloud any difference in T cells.

@Polly Matzinger

Can one first prime the virgin T cells with peptide pulsed DCs (so that, assuming, they present the 'Ag hotdog' to specific virgin T cells) for some time, followed by resting the T cells, and then later using peptide pulsed B7-/- DCs or B cells to induce secondary responses in those specific T cells in vitro?

Because there is no second signal in the restimulation, according to the Laws of lymphotics... only primed T cells specific for the Ag, in this context, will expand and mount memory responses and not unprimed or virgin T cells.

Hi Amarendra,

I like this very interesting discussion.

You get closer to the point:

Virgin means "untouched". No T cell in the circulation will be untouched.

So you need a basic signal for selection and survival of the antigen-specific T cells in the periphery.

So as the thymus may be the primary school, the lymphoid organs holding the circulating immune cells may be the secondary school and the confrontation with antigen in the peripheral organs may be the "real world".

The "real generation" of antigen-specific memory can never be re-capitulated in vitro.

To many factors in vivo simply cannot be found in a petri dish.

This is why we in the end need animal experiments.

So it is correct to speak of re-stimulation or secondary responses.

Why don't you :

Prime your T cells in vitro and compare expansion/cytokine secrection/lytic function after transfer in vivo vs. culture in vitro?

This holds may options.

If you are willing to disuss further...........

Hi, Thilo,

Thank you for your reply. I agree with your argument that won't be any naive T cells in the circulation as it is virtually impossible for a T cell to be in the circulation without untouched. However, I used the term 'virgin' to denote those T cells that are yet to receive a triggering signal which is beyond their activation threshold. Similarly, I used the term 'memory T cell' to denote the ability of a T cell to respond to signal-1 in the absence of signal-2.

I also agree with you that T cell responses are based on the tissue microenvironment and I kind of expected this kind of argument regarding priming T cells in vitro and compare the responses in vivo. This is a fascinating question to check. But, I guess, a T cell would generate the same functional responses second time, which are comparable to the primary response, only when the stimulatory conditions are the same. Thereofore, I wonder if reverse is true, that is... whether it is possible to recapitulate in vitro the same quality and diversity of responses a T cell generate that has been initially primed in vivo with a given Ag peptide.

Cheers

you may recapitulate parts of the re-stimulation in princilpe

yet, it will only minic a very small part of the "true" recall responseb in vivo

how do you define "menory"?

@Thilo

Exactly. The same applies to restimulation of T cells in vivo that were initially primed with the same Ag in vitro in my opinion. Even both events take place in vivo, I wonder, whether a memory T cell recapitulates the same primary response if the location of restimulation is different from the location of priming.

This possible ambiguity in the secondary responses makes it difficult to answer your question ''how do you define 'memory'?'' Perhaps a T cell 'memorizes' that it has already been primed (and therefore, does not look for the signal-2) during the course of subsequent immune responses. In other words, T cell memory is T cell remembering that it has already been received signal-2 during priming and made a productive primary response.

Any other thoughts?