Immune Dysfunctions of Cultured T Lymphocytes Are Due to a Preferential Expansion of CD4+CD25+ Suppressor Cells.

We investigated the causes of the altered functionality of T-cells cultured under conditions designed for cell and gene therapy and the strategies to prevent their defects. We first showed that human T-cells cultured for 6-days with anti-CD3 ± anti-CD28 antibodies and interleukin-2 presented a 50% decrease of their proliferative responses to allogeneic or recall antigens. Similarly, day-6 cultured murine T-cells completely lost their capacity to reject allogeneic skin grafts and to provoke graft versus host disease (GVHD) when infused into irradiated semi-allogeneic mice. Interestingly, injection of higher amount of cultured T-cells restored GVHD induction. Moreover, depletion of CD25+ cells prior to T-cell cultures can prevent these deficiencies both in mice and human. Therefore, we demonstrated that culture conditions used for T-cell therapy preferentially activated and expanded regulatory T-cells (Treg) and thus, we showed that dividing cells sorted from T-cell cultures strongly suppressed the proliferation of autologous T-cells in response to allogeneic stimulation. An increased detection of Foxp3 at mRNA and protein levels in the cultures confirmed the Treg expansion. Overall, we demonstrate that T-cell cultures promote Treg expansion over effector T-cells, leading to deleterious immune functions, and that this imbalance can be prevented by an initial depletion of CD25+ cells.