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Mesenchymal stromal cells (MSCs) have been shown to have immunosuppressive effects in vitro. To test the hypothesis that these effects can be harnessed to prevent graft-versus-host disease (GVHD) and graft rejection after hematopoietic cell transplantation (HCT), we administered a combination of 3 different immortalized marrow-derived MSC lines (15-30 × 106 MSCs/kg/day, 2-5 times/week) or third-party primary MSC (1.0 × 106 MSCs/kg/day, 3 times/week) to canine recipients (n = 15) of dog leukocyte antigen–haploidentical marrow grafts prepared with 9.2 Gy of total body irradiation. Additional pharmacological immunosuppression was not given after HCT. Before their in vivo use, the MSC products were shown to suppress alloantigen-induced T cell proliferation in a dose-dependent, major histocompatibility complex–unrestricted, and cell contact–independent fashion in vitro. Among 14 evaluable dogs, 7 (50%) rejected their grafts and 7 engrafted, with ensuing rapidly fatal acute GVHD (50%). These observations were not statistically different from outcomes obtained with historical controls (n = 11) not given MSC infusions (P = .69). Thus, survival curves for MSC-treated dogs and controls were virtually superimposable (median survival, 18 vs 15 days, respectively). Finally, outcomes of dogs given primary MSCs (n = 3) did not appear to be different from those given clonal MSCs (n = 12). In conclusion, our data fail to demonstrate MSC-mediated protection against GVHD and allograft rejection in this model.

Effects of race or ethnicity on survival after high-dose chemoradiation followed by stem cell transplantation (SCT) have not been thoroughly evaluated. We analyzed survival according to racial/ethnic categories for 3587 consecutive patients who had SCT at a single US institution between July 1992 and December 2000. Among 1366 patients who received autologous SCT, race or ethnicity was not significantly associated with survival. In contrast, among 2221 patients who received allogeneic SCT from HLA-matched unrelated or sibling donors, blacks had a significantly greater mortality than whites (unadjusted hazard ratio, 1.65; 95% confidence interval, 1.21–2.25). Mortality among other racial or ethnic groups was not significantly different from that among whites. The greater mortality hazard among blacks persisted after controlling for donor type, pretransplantation risk category, patient age, donor/patient sex, and cytomegalovirus exposure (hazard ratio, 1.71; 95% confidence interval, 1.25–2.34). SCT from both HLA-matched unrelated and HLA-identical sibling donors was associated with more severe acute graft-versus-host disease and higher nonrelapse mortality among blacks compared with whites. Furthermore, blacks who received SCT for chronic myeloid leukemia had longer diagnosis-to-transplantation intervals than whites. A matched-cohort analysis showed that the higher mortality among blacks could not be explained by obvious socioeconomic differences. The higher incidence of severe graft-versus-host disease among blacks compared with whites, both with HLA-identical sibling donors, might be related to yet-unidentified "immune-enhancing" genetic polymorphisms. We cannot exclude the possibility that the increased mortality risk among blacks after discharge from the transplant center might in part be related to unidentified sociocultural differences that influence medical care.

A multivariate analysis of 121 dogs conditioned with 200, 100, or 50 cGy of total body irradiation (TBI) followed by hematopoietic stem cell transplantation from matched littermates showed that TBI dose was the only factor examined that was statistically significantly associated with the percentage of donor myeloid engraftment in stable long-term chimeras (P = .008). To understand the direct effects of low-dose irradiation on hematopoietic stem/progenitor cells, nonirradiated and irradiated human CD34+ cells were evaluated for competitive repopulating ability in nonobese diabetic/severe combined immunodeficiency beta2m−/− mice. As expected, the results showed a radiation dose-dependent loss of competitive repopulating ability. Flow cytometric analysis indicated that, within a viable cell gate, there was reduced expression of P-selectin glycoprotein ligand-1 and L selectin on irradiated compared with nonirradiated CD34+ cells; this suggests that irradiated stem/progenitor cells may be compromised in their ability to home to or interact with the marrow microenvironment. However, the CD34+/P-selectin glycoprotein ligand-1 dim cells also showed activation of caspase-3, indicating that they were destined to die. These results suggest that the TBI dose determines the degree of myeloid engraftment by compromising the resident stem/progenitor cell compartment.

Mesenchymal stromal cells (MSCs) expanded ex vivo from aspirated marrow, have been used clinically with variable success to facilitate repair of infarcted hearts, treat graft versus host disease, and facilitate marrow reconstitution after radiation damage. While it is now generally acknowledged that these benefits are not the result of engraftment and differentiation of MSC into the target tissues, the mechanism by which these beneficial effects are achieved is not clear. We hypothesize that MSCs mediate their effect by activating an endogenous cell population which in turn modulates the immune response and/or homes to damaged tissue and participates in repair. To begin to test this hypothesis immortalized and cloned populations of canine MSC were generated to provide a consistent product for in vivo testing. One line, designated DS-1, has been evaluated in vivo by infusion into two normal dogs. Blood samples were taken pre infusion, immediately following infusion and at 1, 6, 24, 48, 72, 96 hours, and 7, 14, 21, and 28 days post infusion. Following infusion there was no consistent change in the number of WBC, however by day 3 there was a marked decrease in the % of CD3+ cells expressing FOXP3 and TGFβ in the blood, which did not recover to pre-infusion levels during the period of observation. At autopsy there was an increased number of these cells in the lymph nodes and spleen, whereas there was an overall decrease in the number of TH1 cells in these tissues. Quantitative RT- PCR analysis of cDNA prepared from blood mononuclear cells indicated an upregulation in the expression of CD133, Tie-2, and MARCO between 1–24 hours post infusion, and an increase in LOX1/OLR1 between 2–4 days. However the % of monocytes and the expression levels of CD14, CD68, CD45, and CD105/Endoglin were constant at all time points. Samples taken at 6 hours, 4 and 7 days post infusion were also analyzed for the presence of DS-1 cells by PCR and in vitro out growth assays. Results indicated that the DS-1cells were detectable up to 6 hours post infusion, but not thereafter. Adherent cells grown from blood mononuclear cells at days 4 and 7, displayed macrophage and endothelial cell morphologies. RT-PCR analysis of these cultures detected expression of macrophage associated markers CD14+/CD68+/MARCO+/LOX1+, as well as endothelial cell associated markers CD34+/CD144/VECAD+. These data indicate that a single infusion of DS-1 cells results in activation of circulating monocytes and a shift of regulatory T cells from the periphery to lymph nodes and spleen which persists for at least 28 days. We speculate that these changes may contribute to the immunomodulatory effects reported for some preparations of MSC.

Stable mixed hematopoietic chimerism has been consistently established in dogs who were mildly immunosuppressed by 200 cGy of total body irradiation (TBI) before undergoing dog leukocyte antigen (DLA)-identical bone marrow (BM) transplantation and who received a brief course of immunosuppression with mycophenolate mofetil (28 days) and cyclosporine (35 days) after transplantation. However, when TBI was reduced from 200 to 100 cGy, grafts were nearly uniformly rejected within 3-12 weeks. Here, we asked whether stable engraftment could be accomplished after a suboptimal dose of 100 cGy TBI with host immunosuppression enhanced by donor-derived mesenchymal stromal cells (MSCs) given after transplantation. MSCs were cultured from BM cells and evaluated in vitro for antigen expression. They showed profound immunosuppressive properties in mixed lymphocyte reactions (MLRs) in a cell dose-dependent manner not restricted by DLA. MSC and lymphocyte contact was not required, indicating that immunosuppression was mediated by soluble factors. Prostaglandin E2 was increased in culture supernatant when MSCs were cocultured in MLRs. The addition of indomethacin restored lymphocyte proliferation in cultures containing MSCs. MSCs expressed CD10, CD13, CD29, CD44, CD73/SH-3, CD90/Thy-1, and CD106/VCAM-1. For in vivo studies, MSCs were injected on the day of BM grafting and on day 35, the day of discontinuation of posttransplantation cyclosporine. MSCs derived from the respective BM donors failed to avert BM graft rejection in 4 dogs who received DLA-identical grafts after nonmyeloablative conditioning with 100 cGy TBI in a time course not significantly different from that of control dogs not given MSCs. Although the MSCs displayed in vitro characteristics similar to those reported for MSCs from other species, their immunosuppressive qualities failed to sustain stable BM engraftment in vivo in this canine model.

The application of ex vivo expansion to cell products pharmacologically purged in vitro may provide sufficient numbers of cells for rapid engraftment in a product with reduced tumor burden. To pursue this possibility we evaluated the effect of 4-hydroperoxycyclophosphamide (4-HC) treatment on granulocyte colony-stimulating factor-mobilized peripheral blood stem cells (G-PBSC) and their subsequent expansion potential. A small number of G-PBSC CD34+ cells are resistant to 4-HC and are phenotypically and functionally immature. 4-HC-resistant G-PBSC cells are CD34+ bright, CD38+/-, DR(lo), CD13(lo), CD33-, CD71-, and rhodamine dull. In six experiments, treating G-PBSC with 60 microg/mL of 4-HC at 37 degrees C for 30 minutes reduced the number of colony-forming units (CFUs) per 5000 CD34+ cells by 96.3% (from 1333 +/- 137 to 46.5 +/- 11). This purging also reduced the frequency of 5-week long-term culture initiating cells (LTC-ICs) from 1/39 (range 1/27 to 1/62) to

Emerging evidence suggests that host dendritic cells (DC) initiate and regulate graft-versus-host and graft-versus-tumor reactions after allogeneic hematopoietic cell transplantation (HCT). Even though decades of experimentation in the preclinical canine HCT model have substantially improved our understanding of the biology and safety of HCT in human patients, the in vivo phenotype of potent antigen-presenting cells in dogs is poorly defined. Therefore, peripheral blood leukocytes were obtained from dogs treated with recombinant human Flt3-ligand and phenotypically distinct cell populations, including putative DC, were purified by 4-color flow-cytometry and tested for their stimulatory potential in allogeneic mixed lymphocyte cultures (MLC). Cells characterized by surface expression of CD11c and HLA-DR, and absence of expression of CD14 and DM5, a marker of mature granulocytes, were found to be highly potent stimulators in allogeneic MLC. In contrast, all other immunophenotypically different cell populations tested had either weak or absent allostimulatory potential. Transmission electron microscopy of CD11c+/HLA-DR+/CD14−/DM5− cells revealed the morphology similar to that described for DC in humans and ex vivo-generated canine DC, including long cytoplasmic extensions, discrete lysosomes, and an abundant Golgi apparatus and endoplasmatic reticulum. In summary, CD11c+/HLA-DR+/CD14−/DM5− cells obtained from canine peripheral blood have functional and morphologic characteristics similar to those of human myeloid DC.

Stable mixed donor/host hematopoietic chimerism can be uniformly established in dogs conditioned with 200 cGy TBI before dog leukocyte antigen (DLA)-identical marrow transplantation and immunosuppressed with a short course of mycophenolate mofetil (MMF) and cyclosporine (CSP) after the transplantation. A further decrease in the TBI dose to 100 cGy or the elimination of MMF in this model results in graft rejection. Here we asked whetherthe addition of G-CSF-mobilized peripheral blood mononuclear cells (G-PBMC) to marrow grafts would enhance donor engraftment in dogs conditioned with 100 cGy TBI and given postgrafting immunosuppression with CSP alone. Using this model, 7 of 9 dogs given only marrow cells rejected their grafts within 8 to 17 weeks after transplantation. In contrast, the addition of unmodified G-PBMC to marrow grafts resulted in stable mixed donor/host chimerism in 5 of 8 dogs studied (P = .06). However, addition of the CD3-depleted fraction of G-PBMC, which contained both CD34 cells and CD14 cells, resulted in engraftment in only 1 of 7 recipients. We conclude that adding G-PBMC to marrow grafts replaced the requirement of MMF and 100 cGy of TBI, and that CD3 cells were required to facilitate engraftment of marrow cells in DLA-identical recipients, whereas the additional CD34 cells present in G-PBMC were not sufficient for this effect. Biol Blood Marrow Transplant 2001;7(11):613-9.