On 19 Jun 1995, Isaacs, Cary wrote:
> Our lab has been trying various methods to purify human T cells from peripheral
> blood. We have found columns sold by Pierce to purify consistantly better then
> 90% (based on flow cytometery). However, when we try to stimulate these cells
> with immobilized anti-CD3 and RPMI with 10% FCS, they will not proliferate. The
> PBMC's before the column do proliferate. The T cells also proliferate with PHA.
> Any ideas?
>>> Cary;
There are a number of reasons for these results:
1. Purification of T cells removes antigen-presenting cells required to
give costimulation to T cells along with anti-CD3. In peripheral blood
there is a mixture of CD45RO and CD45RA T cells. The ratio usually
depends on the age of the donor, with older donors having more CD45RO
(memory) T cells that may require less costimulation. But costimulation
they still need.
2. In many cases (e.g., previously primed T cells) costimulation can be
given by the T cells themselves. Thus, by manipulating the density of T
cells in your cultures, you may be able to get adequate IL-2 secretion
and proliferation. Increase the density of your cells in order to get
good cell to cell contact- this should help.
3. Add IL-2 to get the cells going if you have a problem with inadequate
costimulation.
4. Other ways of activating T cells are through allo-antigen or with
ionomycin and phorbol ester.
Laszlo Radvanyi
Ontario Cancer Institute, Princess Margaret Hospital
Toronto, Ontario M4X 1K9
