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).