Treg homing and persistence in NSG mice
Homing of Treg was evaluated using the IVIS technology. Treg expanded
with StM or the IL-7M were labelled with VivoTrack-680 (Perkin Elmer)
and 2x106 of labelled cells were injected
intravenously into NSG mice. In vivo monitoring of labelled cells was
done in a Lumina II IVIS imaging system (Caliper Life Science), by
recording fluorescence at an excitation wavelength of 675 nm and
emission 582 filter of 690-770 nm. Data analysis was done using the
Living Image software v. 4.2 (Caliper Life Sciences). To determine
persistence of Treg in NSG mice, Treg expanded with the StM were
labelled with CFSE and Treg expanded with the IL-7M were labelled with
PBSE, and subsequently co-injected intravenously in NSG mice along with
106 allogenic PBMC used as feeder cells. Treg were
generated from an HLA-A*0201 donor and anti HLA-A*0201-PE (clone BB7.2,
Thermo Fisher) was used to distinguish Treg from feeder PBMC. Blood
samples were collected to detect the presence of Treg generated with the
two expansion protocols. After 14 days mice were sacrificed and the
spleen was collected to detect the presence of Treg generated with the
two expansion protocols.