Graft-versus-host disease suppression by mesenchymal stem cells as determined by cryo-imaging

Abstract

Background & Aim GVHD limits success of allogeneic bone marrow transplantation (alloBMT). Mesenchymal stem cells (MSCs) has been shown to alleviate GVHD, but the biodistribution of the exogenous stem cells and mechanisms behind their protective effects remain to be fully elucidated. We hypothesize that allogeneic T-cells proliferate in secondary lymphoid organs (SLOs) and that therapeutic MSCs home there and suppress T-cell proliferation. Methods, Results & Conclusion We used multi-spectral mouse cryo-imaging (CryoVizTM) to quantitatively evaluate T-cell proliferation, MSC homing, and putative suppression of T-cells by MSCs. Cryo-imaging enables assessments of stem cell biodistribution, homing, and engraftment in whole-mouse with single-cell sensitivity. Briefly, a frozen mouse is sectioned and imaged in a tiled, automated fashion, providing ana¬tomical brightfield and molecular fluorescence, 3D mi-croscopic imaging, to detect labeled cells. Machine learning software accurately detects fluorescent clusters, enabling counting of injected cells. There were four groups of mice: alloBMT (with GVHD), syngeneic BMT (synBME without GVHD), alloBMT+MSCs (GVHD with MSC treatment), and synBMT+MSCs. T-cells were labeled with green-fluorescent CFSE dye, and MSCs were labeled with red-fluorescent quantum dots and tail vein injected. Cryo-imaging was performed on days 3, 4 and 5 post BMT. We examined the bio-distribution of MSCs and T-cells (Fig-A). MSCs homed to SLOs and localized in spleen marginal zone where they could come in contact with T-cells (Fig-B). When cells divide, CFSE dye is distributed to daughter cells and cell signal intensity decreases. Hence reduced CFSE signal intensity in the alloBMT group as compared to the synBMT group is consistent with a greater proliferation of T-cells as a precursor to GVHD (Figs-C-and-D). With MSCs, CFSE signal remained high, indicating a reduction in T-cell proliferation (Fig-E). Similar results were obtained in lymph nodes. Quantitative assays showed homing of about 3,000-4,000 MSCs in alloBMT, significantly more than in synBMT. There was a significant reduction in T-cell proliferation and SLO volumes when the alloBMT group was treated with MSCs. Treatment gave results between alloBMT (with GVHD) and syngeneic BMT (without GVHD). In conclusion, multispectral cryo-imaging allowed us to determine that only a few thousand MSCs could significantly affect T-cell proliferation in SLOs. This is likely one mechanism for MSC suppression of GVHD.