Abstract
Tumor growth factor β (TGF-β)-induced peripheral regulatory T cells (pTreg) are a promising therapeutic cell source that exhibit Foxp3 expression and suppressive functions similar to natural regulatory T cells. Nonetheless, their clinical potential is limited by the instability of Foxp3 expression and T cell exhaustion that occurs during ex vivo expansion. We postulated that mesenchymal stromal cells (MSCs) could enhance the number, function and Foxp3 expression stability of pTregs during IL-2 driven 21 day expansion due to their diverse immunomodulatory properties. In this study, we observed that use of a human bone marrow mesenchymal stromal cells (hBM-MSC) platform significantly enhanced the number of pTreg during IL-2 driven 21 day ex vivo expansion vs. standard suspension culture condition (MSC platform: 80.2 x 106 vs. IL2/media: 39.3 x 106, n=6; p<0.01). Also the number of pTreg expressing a naive phenotype (CD4+CD45RA+ and CD4+CD62L+ ) were significantly increased (CD45RA+; MSC platform: 74.4 ± 1.6 x 106 vs. IL2/media: 45.9 ± 2.9 x 106, n=6, p<0.001; CD62L+; MSC platform: 79.1 ± 1.3 x 106 vs. IL2/media: 54.5 ± 2.1 x 106, n=6, p<0.001), as well as stability of Foxp3 expression (IL-2/media: 88.2 ± 1.7% vs. MSC platform: 96.2 ± 1.1%, n=7; p<0.05). In addition, pTreg suppressive function was noted to be more potent during 21 day IL-2 driven ex vivo expansion compared to standard IL-2/media culture condition (MSC platform: 79% vs. media: 35% inhibition of T cell proliferation in 10:1 ratio, n=6; p<0.01). pTreg expanded over a hBM-MSC platform exhibited higher surface CD25, CTLA-4, and ICOS MFI expression (CD25; MSC platform: 1410 vs. Media: 774; p<0.001, CTLA-4; MSC platform: 1084 vs. Media: 318; p<0.001, ICOS; MSC platform: 4386 vs. Media: 2641, p<0.01, n=6). Notably, hBM-MSC enhancement of pTreg ex vivo expansion requires direct cell-cell contact, as Foxp3 expression in pTreg was not enhanced by hBM-MSC conditioned media (CM:73.4 ± 6.8% vs. MSC platform: 96.2 ± 1.0%, p<0.001; and IL2/media: 88.8 ± 1.6% vs. MSC platform: 96.2 ± 1.0%, p<0.01) nor in a trans-well culture experiments (Transwell: 83.4 ± 2.5% vs. IL2/media: 88.8 ± 1.6%; and Transwell: 83.4 ± 2.5% vs. MSC platform: 96.2 ± 1.0%, p<0.01). Importantly, optical sectioning microscopy and flow cytometry revealed that hBM-MSC supports Treg number and function via direct contact-dependent mitochondrial transfer (Figure 1A-B). Cytochalasin B treatment blocked mitochondrial transfer, suggesting that tunneling nanotubes (TNT) facilitate mitochondrial transfer from hBM-MSC to pTreg during IL-2 driven ex vivo expansion (Mock: 2208 ± 122.1 vs. Cyto B: 923.8 ± 89 MFI, n=6, p<0.0001). Moreover, the quantity of ATP (n=6; p<0.01) mitochondrial potential of pTreg (MSC platform: 9010 ± 224.5 vs. media: 7316 ± 122.7 MFI, n=6; p<0.01) were significantly enhanced in pTreg during IL-2 driven ex vivo expansion over a hBM-MSC platform. Taken together, hBM-MSC significantly improves the number, maturation, and function of pTreg during 21 day IL-2 driven ex vivo expansion. We have identified one key mechanism of action of hBM-MSC underlying these favorable effects on pTreg during ex vivo expansion to be mitochondrial transfer via TNT. Notably, these studies identify a novel role of hBM-MSC to overcome current limitations in IL-2/media suspension culture conditions including T cell senescence, and loss of Foxp3 expression.
Disclosures
No relevant conflicts of interest to declare.
Human Bone Marrow Derived Mesenchymal Stromal Cells Enhance the Number and Function of Umbilical Cord Blood Peripheral Tregs during IL-2 Driven Ex Vivo Expansion