We sought to define the effects and underlying mechanisms of human, marrow-derived mesenchymal stromal cells (hMSCs) on graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) activity. Irradiated B6D2F1 mice given C57BL/6 BM and splenic T-cells and treated with hMSCs had reduced systemic GvHD, donor T-cell expansion, and serum TNFα and IFNγ levels. Bioluminescence imaging demonstrated that hMSCs redistributed from lungs to abdominal organs within 72h; and target tissues harvested from hMSC-treated alloBMT mice had less GvHD than untreated controls. Cryo-imaging more precisely revealed that hMSCs preferentially distributed to splenic marginal zones and regulated T-cell expansion in the white pulp. Importantly, hMSCs had no effect on in vitro cytotoxic T-cell activity and preserved potent GvL effects in vivo. Mixed leukocyte cultures containing hMSCs exhibited decreased T-cell proliferation, reduced TNFα, IFNγ, and IL-10, but increased PGE2 levels. Indomethacin and E-prostanoid 2 (EP2) receptor antagonisms both reversed while EP2 agonism restored hMSC-mediated in vitro T-cell suppression, confirming the role for PGE2. Furthermore, cyclo-oxygenase inhibition following alloBMT abrogated the protective effects of hMSCs. Together, our data show that hMSCs preserve GvL activity and attenuate GvHD and reveal that hMSC biodistribute to secondary lymphoid organs wherein they attenuate alloreactive T-cell proliferation likely through PGE2 induction.

With its single cell sensitivity over volumes as large as or larger than a mouse, cryo-imaging enables imaging of stem cell biodistribution, homing, engraftment, and molecular mechanisms. We developed and evaluated a highly automated software tool to detect fluorescently labeled stem cells within very large (~200GB) cryo-imaging datasets. Cell detection steps are: preprocess, remove immaterial regions, spatially filter to create features, identify candidate pixels, classify pixels using bagging decision trees, segment cell patches, and perform 3D labeling. There are options for analysis and visualization. To train the classifier, we created synthetic images by placing realistic digital cell models onto cryo-images of control mice devoid of cells. Very good cell detection results were (precision=98.49%, recall=99.97%) for synthetic cryo-images, (precision=97.81%, recall=97.71%) for manually evaluated, actual cryo-images, and <1% false positives in control mice. An α-multiplier applied to features allows one to correct for experimental variations in cell brightness due to labeling. On dim cells (37% of standard brightness), with correction, we improved recall (49.26%→99.36%) without a significant drop in precision (99.99%→99.75%). With tail vein injection, multipotent adult progenitor cells in a graft-versus-host-disease model in the first days post injection were predominantly found in lung, liver, spleen, and bone marrow. Distribution was not simply related to blood flow. The lung contained clusters of cells while other tissues contained single cells. Our methods provided stem cell distribution anywhere in mouse with single cell sensitivity. Methods should provide a rational means of evaluating dosing, delivery methods, cell enhancements, and mechanisms for therapeutic cells.

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Graft-vs-host disease (GvHD) limits successful outcomes following allogeneic blood and marrow transplantation (allo-BMT). We examined whether the administration of human, bone marrow-derived, multipotent adult progenitor cells (MAPCs™) could regulate experimental GvHD. The immunoregulatory capacity of MAPC cells was evaluated in vivo using established murine GvHD models. Injection of MAPC cells on day +1 (D1) and +4 (D4) significantly reduced T-cell expansion and the numbers of donor-derived, Tumor Necrosis Factor Alpha (TNFα) and Interferon Gamma (IFNγ)-producing, CD4+ and CD8+ cells by D10 compared with untreated controls. These findings were associated with reductions in serum levels of TNFα and IFNγ, intestinal and hepatic inflammation and systemic GvHD as measured by survival and clinical score. Biodistribution studies showed that MAPC cells tracked from the lung and to the liver, spleen, and mesenteric nodes within 24 hours after injection. MAPC cells inhibited mouse T-cell proliferation in vitro and this effect was associated with reduced T-cell activation and inflammatory cytokine secretion and robust increases in the concentrations of Prostaglandin E2 (PGE2) and Transforming Growth Factor Beta (TGFβ). Indomethacin and E-prostanoid 2 (EP2) receptor antagonism both reversed while EP2 agonism restored MAPC cell-mediated in vitro T-cell suppression, confirming the role for PGE2. Furthermore, cyclo-oxygenase inhibition following allo-BMT abrogated the protective effects of MAPC cells. Importantly, MAPC cells had no effect on the generation cytotoxic T lymphocyte activity in vitro, and the administration of MAPC cells in the setting of leukemic challenge resulted in superior leukemia-free survival. Collectively, these data provide valuable information regarding the biodistribution and regulatory capacity of MAPC cells, which may inform future clinical trial design.

Insights regarding the biodistribution and homing of mesenchymal stromal cells (MSCs), as well as their interaction with alloreactive T-cells are critical for understanding how MSCs can regulate graft-versus-host disease (GVHD) following allogeneic (allo) bone marrow transplantation (BMT). We developed novel assays based on 3D, microscopic, cryo-imaging of whole-mouse-sized volumes to assess the therapeutic potential of human MSCs using an established mouse GVHD model. Following infusion, we quantitatively tracked fluorescently labeled, donor-derived, T-cells and third party MSCs in BMT recipients using multispectral cryo-imaging. Specific MSC homing sites were identified in the marginal zones in the spleen and the lymph nodes, where we believe MSC immunomodulation takes place. The number of MSCs found in spleen of the allo BMT recipients was about 200% more than that observed in the syngeneic group. To more carefully define the effects MSCs had on T cell activation and expansion, we developed novel T-cell proliferation assays including secondary lymphoid organ (SLO) enlargement and Carboxyfluoescein succinimidyl ester (CFSE) dilution. As anticipated, significant SLO volume enlargement and CFSE dilution was observed in allo but not syn BMT recipients due to rapid proliferation and expansion of labeled T-cells. MSC treatment markedly attenuated CFSE dilution and volume enlargement of SLO. These assays confirm evidence of potent, in vivo, immunomodulatory properties of MSC following allo BMT. Our innovative platform includes novel methods for tracking cells of interest as well as assessing therapeutic function of MSCs during GVHD induction. Our results support the use of MSCs treatment or prevention of GVHD and illuminate the wider adoption of MSCs as a standard medicinal cell therapy.