In vitro performances of naïve Treg expanded with the StM or the IL-7M
As expanded Treg were shown to undergo a rapid and substantial reduction once infused in patients, we aim to determine the in vitro performances of Treg expanded with the StM or the IL-7M. First, we determined parameters associated to the bio-energetic metabolism that can account for the metabolic fitness of expanded Treg. In vitro Treg expand in the presence of high glucose concentrations and availability of oxygen but these two conditions are not always met in the human body, where glucose concentration is 5.5mM and oxygen availability is highly dependent on the anatomical localization, potentially affecting the Treg capacity to fulfill the metabolic needs once infused in patients. Treg expanded with the IL-7M showed an increased mitochondrial mass (MFI, IQR) compared to Treg expanded with the StM (StM 422, 240-743 vs IL-7M 1221, 1082-1427, p=0.031) as measured using the MitoTraker™ Green dye that accumulates in mitochondria regardless of mitochondrial membrane potential (Figure 4A). A similar mitochondrial membrane potential (ΔψM) was found in the two groups as measured using the MitoTracker® Deep Red FM dye (Figure 4B). To measure glucose uptake capacity we performed a dynamic assay by measuring the uptake of the glucose analog 2NBDG over a 50 minute time window. Data were analysed by comparing the differences in the area under the curve (AUC). Treg generated with the IL-7M showed a greater capacity to accumulate 2NBDG as compared to Treg generated with the StM (AUC; StM 3.5x106 vs IL-7M 4.8x106) (Figure 4C). Corroborating these findings, Treg expanded with the IL-7M produced more lactate that Treg expanded with the StM (StM 182, 133-233 vs IL-7M 367, 295-430, p=0.03) (Figure 4D) suggesting an increased glycolytic rate. These immuno-metabolic signatures are preserved after a resting period of three days in the absence of cytokines and antiCD3CD28 microbeads even if the intensity of signals are reduced accordingly to a lack of growth factors (Suppl Figure 3A). To test the performances of Treg in stress conditions, we performed experiments in which expanded Treg where cultured without cytokines and antiCD3/CD28 microbeads replicating the environmental change that Treg will face once infused in patients. The number of Treg expanded with the IL-7M declined less sharply than the number of Treg expanded with the StM. After three weeks of culture a significantly higher number of Treg expanded with the IL-7M was still viable (absolute number x103; StM: 46, 26-71 vs IL-7M 338, 272-386, p=0.031) (Figure 4E). Notably, when cytokines and beads were added back to the culture, Treg expanded with the IL-7M were also able to recover and re-expand after a second challenge. We next challenged expanded Treg with a direct pro-apoptotic stimulus using an agonistic anti-fas IgM antibody. We found that the percentage of Treg undergoing apoptosis was significantly lower in Treg expanded with the IL-7M (% annexin V+: StM 69.8, 41.9-78.2 vs IL-7M, 35.4, 21.6-39-6, p=0.018) (Figure 4F). As IL-7 was shown to up-regulate the anti-apoptotic molecule Bcl-2, we measured Bcl-2 in Treg expanded with the two methods and compared to Treg-n before expansion (Figure 4G). While expanded Treg showed an up-regulation of Bcl-2 compared to the non-expanded Treg-n, Treg expanded with the IL-7M display a significantly higher expression of Bcl-2 (MFI: StM 464, 320-684 vs IL-7M 892, 510-1142, p=0.043). Finally we measured the length of telomeric DNA after expansion as telomere erosion is associated with cell senescence. As compared to Treg-n, we observed a substantial reduction in telomere length in expanded Treg with both methods, but Treg expanded with the IL-7M showed significant preservation of telomeric DNA compared to Treg expanded with the StM (MFI: StM 729, 456-880 vs IL-7M 1356, 1095-1712, p= 0.0158).