In the online 9/13/05 Blood article titled "Initial depletion of
regulatory T-cells: the missing solution to preserve the immune
functions of T lymphocytes designed for cell-therapy,"
Mesel-Lemoine et al. state:

"Overall our results underline that culture conditions commonly used
for the expansion of T-cells designed for cell/gene therapy favor the
expansion of Treg, which in turn suppress further functionality of
T-cells both in vitro and in vivo. Interestingly, the initial
depletion of CD25+ T-cells before cell activation and/or expansion
proves to be a simple strategy that prevents Treg/effector imbalance
and preserves T-cell functionality."

"Overall, our findings underline that the unexpected expansion of Treg
is responsible for the culture-induced T-cell defects and suggest new
strategies for improving the functions of T-cells designed for cell
and/or gene therapy protocols."

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Blood First Edition Paper, prepublished online September 13, 2005; DOI
10.1182/blood-2005-07-2658.

Initial depletion of regulatory T-cells: the missing solution to
preserve the immune functions of T lymphocytes designed for
cell-therapy

Mariana Mesel-Lemoine, Mustapha Cherai, Sabine Le Gouvello, Maude
Guillot, Virginie Leclercq, David Klatzmann, Veronique Thomas-Vaslin,
and Francois M Lemoine*

CNRS UMR 7087/UPMC, Hopital de la Pitie Salpetriere, Paris, France
CNRS UMR 7087/UPMC, Hopital de la Pitie Salpetriere, Paris, France;
Service de Biotherapies, Hopital de la Pitie Salpetriere, Paris,
France Laboratoire d'Immunologie Biologique, Hopital Henri Mondor,
Creteil, France

* Corresponding author; email: francois.lemoine{at}chups.jussieu.fr.

Abstract: 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 expande regulatory T-cells (Treg). 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.