No abstract

Optimization of T-cell-activation protocols is an important prerequisite for the use of populations of activated, polyclonal T cells for immunotherapeutic purposes. This study compares two activation protocols. Following initial CD3/CD28 activation, naïve and memory subsets of CD8 þ and CD4 þ T cells were either repeatedly stimulated or maintained in medium containing interleukin-2 (IL-2). Initially, activation-induced cell death (AICD) was observed in all subsets. After 2-3 days, death in the cultures maintained in IL-2 only dropped dramatically, while live cells increased logarithmically. Despite intense proliferation, these cells lost the expression of CD25, the a chain of the IL-2 receptor and CD71, the transferrin receptor. Functional blocking of CD25 caused minimal changes in proliferation and survival of these cells as long as IL-2 was present in the medium. Blocking of CD25 in combination with the removal of IL-2 caused rapid death of these cells. Restimulation every 3-4 days led to persistently high levels of AICD and lower live cell counts. Live cells maintained the expression of activation markers and a blastoid phenotype. Initial CD3/CD28 followed by maintenance in IL-2 for 2-3 weeks seems to be the best in vitro T-cell-activation strategy. Signalling through the IL-2 receptor is vital for these cells, despite their downregulation of CD25.

No abstract

Changes in the composition of the T-cell subsets at the end of the cell culture were the results of the activation, and not the suicide gene transduction. The transduced T cells did not express unwanted homing receptors.

These results should impact on protocols for ex vivo activation of T-cells prior to suicide gene transduction.