Homing and survival of Treg expanded with the StM or the IL-7M
in NSG mice
To determine differences in the in vivo homing capacity and survival,
Treg were injected in NSG mice. To determine homing expanded and
IVI-sense-labelled Treg generated with the StM or the IL-7M were
injected in the tail vein of NSG mice and monitored for 48 hours using
the IVIS technology. After injection of Treg expanded with the StM, we
observed an increased signal in the bladder (Figure 5A). As the labeling
dye is excreted with urine we interpreted this signal as a consequence
of increased cell death and release of free dye that accumulate in the
bladder. After injection of Treg expanded with the IL-7M we noticed an
increased signal in the femurs of mice, indicating an improved home
capacity to the bone marrow. Corroborating these findings we observed an
increased expression of CXCR4 in Treg expanded with the IL-7M (Figure
5B). Treg expanded with the IL-7M showed also an increased expression of
CCR7 (also in line with a poorly differentiated phenotype), a similar
but very low expression of CCR2 and a reduced expression of CCR9,
indicating other possible differences in the migratory pattern of Treg
expanded with the StM or the IL-7M. We next determined the overall
persistence of Treg expanded with the StM or the IL-7M by injecting Treg
intra-peritoneally in NSG mice. To distinguish injected Treg from
allogenic PBMC used as feeder cells in the model we selected HLA-A*0201
Treg for expansion and track them in NSG mice using a anti HLA-A*0201
monoclonal antibody (Figure 5C). Treg expanded using the StM or the
IL-7M protocol were co-injected in the same mice and labeled with CFSE
and PBSE respectively for tracking. In peripheral blood we were able to
detect Treg expanded with the IL-7M for approximately 11 days whereas
Treg expanded with the StM were detectable only until day 5 (Figure 5D).
After 14 days from injection (with undetectable circulating Treg) mice
were sacrificed and spleens were removed to detect the presence of
residual Treg. We found that a significantly higher percentage of Treg
expanded with the IL-7M compared to Treg expanded with the StM were
present in the spleen of mice, consistent with a prolonged capacity to
survive in vivo (Figure 5E).