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Osteosarcoma is a high-grade bone-forming neoplasm, with a complex genome. Tumours frequently show chromothripsis, many deletions, translocations and copy number alterations. Alterations in the p53 or Rb pathway are the most common genetic alterations identified in osteosarcoma. Using spontaneously transformed murine mesenchymal stem cells (MSCs) which formed sarcoma after subcutaneous injection into mice, it was previously demonstrated that p53 is most often involved in the transformation towards sarcomas with complex genomics, including osteosarcoma. In the current study, not only loss of p53 but also loss of p16(Ink4a) is shown to be a driver of osteosarcomagenesis: murine MSCs with deficient p15(Ink4b), p16(Ink4a), or p19(Arf) transform earlier compared to wild-type murine MSCs. Furthermore, in a panel of nine spontaneously transformed murine MSCs, alterations in p15(Ink4b), p16(Ink4a), or p19(Arf) were observed in eight out of nine cases. Alterations in the Rb/p16 pathway could indicate that osteosarcoma cells are vulnerable to CDK4/CDK6 inhibitor treatment. Indeed, using two-dimensional (n = 7) and three-dimensional (n = 3) cultures of human osteosarcoma cell lines, it was shown that osteosarcoma cells with defective p16(INK4A) are sensitive to the CDK4/CDK6 inhibitor palbociclib after 72-hour treatment. A tissue microarray analysis of 109 primary tumour biopsies revealed a subset of patients (20-23%) with intact Rb, but defective p16 or overexpression of CDK4 and/or CDK6. These patients might benefit from CDK4/CDK6 inhibition, therefore our results are promising and might be translated to the clinic.Osteosarcoma is a tumour with a highly complex genome, which hampers the identification of driver genes. Using a model of murine mesenchymal stem cells (MSCs) with deficient p15(Ink4b), p16(Ink4a), or p19(Arf) that transform earlier compared to wild-type MSCs, the authors demonstrated that loss of p16(Ink4a) is a driver of osteosarcomagenesis. This can be exploited with a CDK4/CDK6 inhibitor, as osteosarcoma cells showed sensitivity to palbociclib which might be used as a novel therapeutic option.

Mesenchymal chondrosarcomas are rare and highly aggressive sarcomas occurring in bone and soft tissue, with poor overall survival. Bcl-2 expression was previously shown to be upregulated in mesenchymal chondrosarcomas. We here report on a newly derived mesenchymal chondrosarcoma cell line, MCS170, in which we investigated treatment with the BH3 mimetic ABT-737 alone or in combination with conventional chemotherapy as a possible new therapeutic strategy. The presence of the characteristic HEY1-NCOA2 fusion was confirmed in the MCS170 cell line using FISH, RT-PCR, and sequencing. The MCS170 cell line was treated with ABT-737 alone or in combination with doxorubicin or cisplatin. Cell viability and proliferation was determined using WST-1 viability assays and the xCELLigence system. Expression of Bcl-2 family members was studied using immunohistochemistry. Apoptosis was determined using the caspase-glo 3/7 assay and western blot for PARP cleavage. The MCS170 cell line was sensitive to doxorubicin treatment with an IC50 of 0.09 μM after 72 h, but more resistant to cisplatin treatment with an IC50 of 4.5 μM after 72 h. Cells showed little sensitivity toward ABT-737 with an IC50 of 1.8 μM after 72 h. Combination treatments demonstrated ABT-737 synergism with cisplatin as well as doxorubicin as shown by induction of apoptosis and reduction in cell proliferation. Restoration of the apoptotic machinery by inhibition of Bcl-2 family members sensitizes MCS170 mesenchymal chondrosarcoma cells to conventional chemotherapy. This indicates that combining the inhibition of Bcl-2 family members with conventional chemotherapy can be a possible therapeutic strategy for patients with mesenchymal chondrosarcoma.

Objective Umbilical cord blood (UCB) is considered as an attractive alternative source of hematopoietic stem cells for allogeneic stem cell transplantations in patients who lack human leukocyte antigen (HLA)-matched donors. However, the low cell dose adversely affects hematopoietic recovery and therefore limits application of UCB transplantation in adults. Transplantation of multiple UCB units could be a strategy to overcome cell dose limitations. Materials and Methods To investigate the effect of double cord transplantation, nonobese diabetic/severe combined immunodeficient mice were transplanted with human hematopoietic progenitor cells (CD34 + ) derived from two UCB units with HLA disparity. Human cell engraftment and donor origin was determined by flow cytometry. Results Double CB transplantation resulted in increased engraftment levels in the bone marrow and peripheral blood in comparison with recipients of a single unit. Because this effect could be due to the higher cell dose (2.10 5 vs 1.10 5 cells), double CB transplantation was compared with single units containing equal cell numbers (2.10 5 ). In some cases, engraftment levels in recipients of single units containing 2.10 5 cells were significantly higher than after transplantation of 1.10 5 cells. These engraftment levels were similar to those observed after double CB transplantation. Chimerism analysis indicated that increased engraftment in recipients of two units was predominantly derived from one unit, whereas in other cases the contribution of the two units was similar. Conclusion These results indicate that engraftment may be enhanced by addition of a second unrelated CB that might be attributed to a cell dose effect or due to a graft-facilitating effect.

Objective Mesenchymal stem cells (MSC) have been implicated as playing an important role in hematopoietic stem cell engraftment. We identified and characterized a new population of MSC derived from human fetal lung. In cotransplantation experiments, we examined the homing of MSC as well as the effect on engraftment of human umbilical cord blood (UCB)-derived CD34 + cells in NOD/SCID mice. Materials and Methods Culture-expanded fetal lung-derived CD34 + cells were characterized by immune phenotyping and cultured under conditions promoting differentiation to osteoblasts or adipocytes. Irradiated (3.5 Gy) NOD/SCID mice (n = 51) were transplanted intravenously with 0.03 to 1.0 × 10 6 UCB CD34 + cells in the presence or absence of 1 × 10 6 culture-expanded fetal lung-derived MSC, irradiated CD34 − cells, B cells, or with cultured MSC only. Results Culture-expanded fetal lung CD34 + cells were identified as MSC based on phenotype (CD105 + , SH3 + , SH4 + , CD160 + ) and their multilineage potential. Cotransplantation of low doses of UCB CD34 + cells and MSC resulted in a three-fold to four-fold increase in bone marrow engraftment after 6 weeks, whereas no such effect was observed after cotransplantation of irradiated CD34 − or B cells. Homing experiments indicated the presence of MSC in the lung, but not in the bone marrow, of NOD/SCID mice. Conclusions We identified a population of MSC derived from human fetal lung. Upon cotransplantation, MSC, but not irradiated CD34 − or B cells, promote engraftment of UCB CD34 + cells in bone marrow, spleen, and blood by mechanisms that may not require homing of MSC to the bone marrow.

CD8+ T cell-depleted (TCD) donor lymphocyte infusion (DLI) after TCD allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with a reduced risk of graft-versus-host disease (GVHD) while preserving conversion to donor hematopoiesis and antitumor immunity, providing a rationale for exploring CD4+ T cell-based immunotherapy for hematologic malignancies. Here, we analyzed the clinical course and specificity of T cell immune responses in 2 patients with acute myeloid leukemia (AML) who converted to full-donor chimerism but developed severe acute GVHD after prophylactic CD4+ DLI after 10/10-HLA-matched, but HLA-DPB1-mismatched TCD-alloSCT. Clonal analysis of activated T cells isolated during GVHD demonstrated allo-reactivity exerted by CD4+ T cells directed against patient-mismatched HLA-DPB1 molecules on hematopoietic cells and skin-derived fibroblasts only when cultured under inflammatory conditions. At the time of CD4+ DLI, both patients contained residual patient-derived T cells, including cytomegalovirus (CMV)-specific T cells as a result of CMV reactivations. Once activated by CMV antigens, these CMV-specific T cells could stimulate HLA-DPB1-specific CD4+ T cells, which in turn could target nonhematopoietic tissues in GVHD. In conclusion, our data demonstrate that GVHD after HLA-DPB1-mismatched CD4+ DLI can be mediated by allo-reactive HLA-DPB1-directed CD4+ T cells and that ongoing viral infections inducing HLA class II expression on nonhematopoietic cells may increase the likelihood of GVHD development. This trial is registered at http://www.controlled-trials.com/ISRCTN51398568/LUMC as #51398568.

Abstract 4031 Following HLA-matched allogeneic stem cell transplantation (SCT) donor T cells may recognize polymorphic antigens specifically expressed on cells of the recipient, designated as minor histocompatibility antigens (MiHA). It has been hypothesized that hematopoiesis-restricted MiHA may give rise to a specific graft versus leukemia (GvL) effect without graft versus host disease (GvHD), whereas ubiquitously expressed MiHA can be targets for both GvL and GvHD. We and others have observed that during an overt GvL response, associated with the occurrence of T cells specific for hematopoiesis-restricted MiHA, may coincide with limited GvHD. Therefore, we investigated whether allo-reactive T cells recognizing hematopoiesis-restricted MiHA on leukemic cells may induce collateral damage to surrounding non-hematopoietic tissues. To test this, we stimulated MiHA-specific cytotoxic T lymphocytes (CTLs) in vitro with MiHA positive hematopoietic targets and analyzed whether this resulted in lysis of surrounding MiHA negative primary human fibroblasts seeded in the same well. Using chromium release assays, quantitative FACS analysis and fluorescence microscopy, we demonstrated that MiHA-specific CTLs (CD8 n=3, CD4 n=2) and allo-reactive CTLs upon proper activation by MiHA-positive hematopoietic stimulator cells (EBV-LCL/ DCs), induced 80% (60–100%) bystander cytotoxicity to the surrounding fibroblasts at a 5/1 T cell/fibroblast-ratio within 24 hrs. T cell receptor (TCR)-specific T cell activation by the hematopoietic cells was pivotal for the induction of this collateral damage since no bystander kill was observed when MiHA-specific CTLs were exposed to MiHA negative stimulators. Next, we investigated the influence of the strength of T cell activation by stimulating the CTLs with MiHA-negative stimulator cells loaded with increasing concentrations of the relevant peptide resulting in increasing T cell activation measured by IFNg ELISA and expression of the T cell activation marker CD137. We demonstrated that this increase in the strength of T cell activation is correlated with an increased severity of collateral damage. Moreover, we showed also that T cell activation in a cell free system using PHA (0,5ug/ml) or antiCD3/CD28 beads resulted in induction of collateral damage of the surrounding non-hematopoietic cells illustrating that the MiHA-negative fibroblasts are not attacked due to cross-presentation of HLA/peptide complexes from apoptotic hematopoietic cells. Next, we further investigated the prerequisites for collateral damage induction to occur. Using a transwell system, we demonstrated that direct T cell-fibroblast interaction was required. Furthermore, ICAM-1 expression on the non-hematopoietic targets, induced by pro-inflammatory cytokines which were secreted by the activated CTLs, was demonstrated to be crucial for strong T cell-fibroblast interaction. To investigate the contribution of the different T cell effector mechanisms, we retrovirally overexpressed the anti-apoptotic proteins c-FLIP, Bcl-2 or PI-9 in the fibroblasts, thereby inhibiting the death receptor-, mitochondrial-, or granzyme-B (GzB)-mediated pathways, respectively. High expression of c-FLIP or Bcl-2 did not inhibit induction of collateral damage. In contrast, increased PI-9 expression in the fibroblasts partially inhibited induction of collateral damage. Moreover, blockade of Ca2+-dependent secretion of perforin by EGTA, could also inhibit collateral damage suggesting that the main killing pathway of collateral damage is via release of GzB- and perforin-containing granules by the T cells upon TCR-triggering. Misdirection of the release of cytotoxic granules towards the MiHA negative non-hematopoietic targets may induce collateral damage. In conclusion, these data suggest that hematopoiesis-restricted T cells actively participating in an overt GvL response can induce GvHD by collateral damage to MiHA-negative non-hematopoietic targets by misdirection of their cytotoxic execution machinery when they are in close proximity of the non-hematopoietic tissue. Disclosures: No relevant conflicts of interest to declare.

Mesenchymal stem cells (MSCs) are not only able to evade the immune system, but they have also been demonstrated to exert profound immunosuppressive properties on T cell proliferation. However, their effect on the initiators of the immune response, the dendritic cells (DCs), are relatively unknown. In the present study, the effects of human MSCs on the differentiation and function of both CD34+-derived DCs and monocyte-derived DCs were investigated. The presence of MSCs during differentiation blocked the differentiation of CD14+CD1a− precursors into dermal/interstitial DCs, without affecting the generation of CD1a+ Langerhans cells. In line with these observations, MSCs also completely prevented the generation of immature DCs from monocytes. The inhibitory effect of MSCs on DC differentiation was dose dependent and resulted in both phenotypical and functional modifications, as demonstrated by a reduced expression of costimulatory molecules and hampered capacity to stimulate naive T cell proliferation. The inhibitory effect of MSCs was mediated via soluble factors. Taken together, these data demonstrate that MSCs, next to the antiproliferative effect on T cells, have a profound inhibitory effect on the generation and function of both CD34+-derived and monocyte-derived DCs, indicating that MSCs are able to modulate immune responses at multiple levels.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that have emerged as a promising tool for clinical application. Further clinical interest has been raised by the observation that MSCs are immunoprivileged and, more important, display immunosuppressive capacities. These properties may be of therapeutic value in allogeneic transplantation to prevent graft rejection and to prevent and treat graft-versus-host disease. In the present study, we examined the in vivo immunomodulatory properties of MSCs in murine models of allogeneic bone marrow (BM) transplantation. Sublethally irradiated recipients received allogeneic BM with or without host or donor MSCs. The addition of host MSCs significantly enhanced the long-term engraftment associated with tolerance to host and donor antigens. However, the infusion of donor MSCs was associated with significantly increased rejection of allogeneic donor BM cells. Moreover, we showed that the injection of merely allogeneic donor MSCs in naive mice was sufficient to induce a memory T-cell response. Although the observed engraftment-promoting effects of host MSCs in vivo support the therapeutic potential of MSCs, our results also indicate that allogeneic MSCs are not intrinsically immunoprivileged and that under appropriate conditions, allogeneic MSCs induce a memory T-cell response resulting in rejection of an allogeneic stem cell graft.

Objective Previously, we have found that human culture-expanded fetal lung–derived mesenchymal stem cells (MSC) promote the engraftment of umbilical cord blood (UCB)-derived CD34 + cells. The high frequency of MSC in fetal lung allowed us to study whether this represented a biological feature of these cells or a property that was acquired during expansion in culture. Materials and methods Irradiated NOD/SCID mice (n=80) were transplanted with 0.1×10 6 UCB CD34 + cells in the presence or absence of 10 6 primary nonexpanded or culture-expanded fetal lung, liver, or BM CD45 − cells, or with nonexpanded fetal lung liver or BM CD45 − cells only. Results In comparison with transplantation of UCB CD34 + cells only, cotransplantation of UCB CD34 + cells and primary fetal lung or BM CD45 − cells resulted in a significantly higher level of engraftment (% hCD45 + cells) in BM, PB, and spleen. In addition, primary mesenchymal cells derived from adult BM had a similar promoting effect. The engraftment-enhancing effect was similar to that of culture-expanded fetal lung and BM MSC. Primary mesenchymal cells, but not culture-expanded MSC, were detected in recipient mice, suggesting that the primary cells were able to home and that this capacity was lost after expansion. Conclusion These results show that primary mesenchymal cells from fetal lung and BM promote the engraftment of UCB-derived CD34 + cells to a similar degree as culture-expanded MSC, indicating that it reflects a biological property of primary MSC that is preserved during expansion in culture.

Abstract 3552 Poster Board III-489 Graft versus host disease (GvHD) after allogeneic stem cell transplantation (SCT) may occur when donor T cells respond to antigens specifically expressed on cells of the recipient, such as minor histocompatibility antigens (mHags). It has been hypothesized that ubiquitously expressed mHags, like HY and ADIR-1F, may be targets for both GvHD and graft versus leukemia (GvL), whereas hematopoiesis-restricted mHags, such as HA-1, may give rise to a more specific GvL effect without GvHD. However, it has been reported that mismatching for HA-1 is correlated with the development of GvHD. In addition, there is clinical evidence that the presence of a high tumor load leading to a profound GvL reaction may be associated with the induction of GvHD. To explain these observations, we analyzed whether T cells directed against hematopoiesis-restricted antigens, like HA-1, are able to contribute to destruction of the surrounding non-hematopoietic tissues in the effector phase of the immune response. First, we investigated whether HA-1 specific T cells could directly attack non-hematopoietic tissues, such as human primary skin fibroblasts from HLA-A2+, HA-1+ individuals using a 51Cr-release assay. In accordance with the absent gene expression of HA-1 in fibroblasts, no direct antigen-specific kill of the fibroblasts was seen. In contrast, exposure of ADIR-1F mHag positive fibroblasts to ADIR-specific CTLs resulted in 70% lysis after 20 hours. Even in a more inflammatory environment, induced by pretreatment with interferon gamma (IFNg), primary human fibroblasts from HLA-A2+, HA-1+ individuals were not directly killed by HA-1 specific CTLs. However, based on the clinical observation that the induction of a profound GvL effect in patients with a high tumor load correlated with the induction of GvHD, we hypothesized that these hematopoiesis-restricted T cells may have the capacity to induce collateral damage to surrounding non-hematopoietic tissues during an ongoing profound immune response against the hematopoietic target cells. To test this hypothesis, we developed an in vitro system in which monolayers of 51Cr-labeled primary human fibroblasts from HLA-A2+, HA-1+ individuals were co-cultured with HA-1-specific CTLs in the presence or absence of HA-1-expressing hematopoietic targets (EBV-LCL) for 4, 8 and 20hrs. Activation of HA-1-specific T cells by the EBV-LCL was shown to be dose dependent and was found to be optimal at stimulator/responder (S/R) ratios higher than 1/1 as measured by their specific IFNg production. Despite optimal activation of the HA-1-specific CTLs by the EBV-LCL (S/R ratio 10/1), initially no collateral damage to surrounding fibroblast was observed. However, under pro-inflammatory conditions, mimicked by the treatment of the fibroblasts with IFNg, HA-1-specific CTLs optimally activated by the HA-1+ EBV-LCL which were seeded between the fibroblasts (S/R ratio 10/1), exerted 40% (25-60%) bystander cytotoxicity to the surrounding fibroblasts at a 10/1 T cell/fibroblast-ratio after 20 hrs. Interestingly, there was a strict correlation between the amount of collateral damage to the surrounding fibroblasts and the strength of the T cell activation by the hematopoietic cells expressing the relevant target antigen, being optimal at S/R ratios higher than 1/1, possibly reflecting the clinical situation of a GVL response in the presence of a high tumor load. Similarly, the magnitude of the immune response, reflected by the number of T cells, was associated with the amount of collateral damage, since no collateral damage could be observed at CTL/fibroblast ratios below 3/1. In conclusion, these data indicate that a profound immune response against hematopoietic targets, mediated by T cells directed against hematopoiesis-restricted antigens, may result in the induction of GvHD via collateral damage to the surrounding non-hematopoietic tissues without the necessity for these tissues to express the specific target antigen. Disclosures: No relevant conflicts of interest to declare.

Abstract 2454 Poster Board II-431 Donor T cells specific for minor histocompatibility antigens (mHags) contribute to graft-versus-leukemia (GvL) reactivity and graft versus host disease (GvHD) following HLA-matched stem cell transplantation. Hematopoiesis-restricted mHags may give rise to a more specific GvL effect without GvHD, whereas ubiquitously expressed mHags can be targets for both GvHD and GvL. Interestingly, high frequencies of circulating T cells specific for ubiquitously expressed mHags (e.g. male-specific epitopes, LB-ADIR-1F) have been detected in patients showing strong GvL with no or limited GvHD. Although both hematopoietic and non-hematopoietic cells express the proteins encoding these mHags, the immune effector response in these patients appears to be skewed towards hematopoietic cells. Therefore, we hypothesized that non-hematopoietic tissues are relatively unsusceptible to the cytotoxic effect of these mHag-specific T cells. To test this hypothesis, we developed an in vitro system in which HLA-A2+, ADIR+ primary human fibroblasts were exposed to mHag LB-ADIR-1F or allo-A2 specific CD8+ T cells. ADIR+ EBV-LCL were used as a control for hematopoietic cells. CTL-mediated cytotoxicity was measured using 4, 8 and 20 hours chromium release assays. Whereas all EBV-LCL were killed within 4 hours, 10% lysis of fibroblasts was found after 4 hours, only gradually increasing to 70% lysis after 20 hours in the continuous presence of the CTLs at 10/1 effector/target (E/T) ratios. To analyze whether this inefficient killing was due to low antigen expression by the human primary fibroblasts, we examined cytotoxicity against targets loaded with increasing concentrations of ADIR-peptide. Exogenous loading of fibroblasts with saturating concentrations (10-6M) of ADIR-peptide did not increase the sensitivity to T cell mediated cell death. To exclude that the observed inferior cell killing was due to the number of T cells available for engaging the fibroblasts, E/T ratios were increased to >100/1, but this did not change the poor susceptibility of the fibroblasts to CTL-mediated killing. Since apparently inefficient elimination of non-hematopoietic targets was found despite proper expression of the relevant antigen and the presence of functional T cells, we investigated whether insufficient engagement of T cells during contact with the fibroblasts was the cause of the impaired killing. To analyze this, kinetics of T cell degranulation upon target encounter was measured by CD107a, perforin and granzyme B expression. Although the onset of T cell activation was already seen after 2 hours, stimulation with fibroblasts resulted in degranulation in only a proportion of the T cells (50-60%) after 4hrs, resulting in full activation only after prolonged co-culture. In contrast, stimulation with EBV-LCL resulted in rapid, complete, and profound degranulation (100% of T cells within 2hrs). In concordance with this, T cell activation, as measured by the production of interferon gamma (IFNg) after stimulation with the fibroblasts, was suboptimal as compared to stimulation with EBV-LCL. These data suggest formation of an initial low avidity interaction with fibroblasts under steady state conditions, possibly only leading to efficient T cell engagement and fibroblast killing after a prolonged period of co-culture, resulting in the formation of a local pro-inflammatory environment. To confirm whether susceptibility to mHag-specific T cells was increased under pro-inflammatory conditions, we pre-treated fibroblasts with IFNg. Analysis of the expression of a panel of known adhesion and co-stimulatory molecules revealed that IFNg treatment resulted in a 2-5 fold upregulation of expression of HLA class I, HLA class II and ICAM-1, leading to a more uniform and profound T cell activation. This increased T cell activation resulted in more efficient direct fibroblast killing. In conclusion, despite proper expression of the relevant target antigens, low-avidity interaction between effector T cells and non-hematopoietic tissues results in minimal damage under non-inflammatory conditions. Disclosures: No relevant conflicts of interest to declare.

Mesenchymal stem cells (MSCs) have been demonstrated to exert profound immunosuppressive properties on T cell proliferation. However, their effect on the initiators of the immune response, the dendritic cells (DCs), are relatively unknown. In the present study, the effects of MSCs on the differentiation and function of both monocyte-derived DCs and CD34+-derived DCs were investigated. Monocytes (CD1a-CD14+) were obtained from PB and were cultured with IL-4 and GM-CSF to induce differentiation into CD14-CD1a+ immature DCs. CD34+ hematopoietic progenitor cells were isolated from umbilical cord blood samples and cultured in the presence of GM-CSF, TNF-a, and SCF to generate Langerhans cells, which differentiate directly into CD1a+ DCs, and dermal/interstitial DCs, which differentiate via an intermediate CD14+CD1a- phenotype into CD14-CD1a+ DCs. MSCs were generated from fetal lung tissue as reported previously (Exp. Hematol.2002; 30: 870–878). The phenotype (CD1a, CD14, CD80, CD86, CD83, HLA-DR, CD40) of the cells was analyzed by flow cytometry; cytokine production (IL-12, TNF-α) was examined by enzyme-linked immunosorbent assay (ELISA) and T cell stimulatory capacity was determined by a mixed lymphocyte reaction (MLR). The presence of MSCs during the complete differentiation period completely prevented the generation of immature DCs (CD1a+CD14-) from monocytes in a dose-dependent manner. MSCs in the upper wells of a transwell culture system inhibited the differentiation of monocytes in the lower wells, indicating that the suppressive effect of MSCs was mediated via soluble factors. The inhibitory effect of MSCs on the differentiation of DCs was partially prevented by the addition of neutralizing antibodies to IL-6 and M-CSF, indicating the involvement of these cytokines. Upon removal of MSCs cultured in a transwell after 48h, differentiation of monocytes towards DCs was restored, indicating that the suppressive effect of MSCs was reversible. DCs generated in the presence of MSCs were unresponsive to signals inducing maturation (CD40 ligand, lipopolysaccharide), as demonstrated by the absence of CD83, CD80, CD86 and HLA-DR upregulation and the decreased production of the inflammatory cytokines TNF-α (76%) and IL-12 (79%). In addition, the T cell stimulatory capacity of mature DCs generated in the presence of MSCs was strongly reduced. MSCs also inhibited the generation of DCs from CD34+ progenitor cells by blocking the differentiation of CD14+CD1a- precursors into dermal/interstitial DCs, without affecting the generation of CD1a+ Langerhans cells. The inhibitory effect of MSCs on CD34+ cell differentiation was dose-dependent and resulted in both phenotypical and functional modifications, as demonstrated by a reduced expression of costimulatory molecules (CD80, CD86) and CD83, and hampered capacity to stimulate naïve T-cell proliferation (50,112 ± 1,305 cpm versus 20,412 ± 1,593 cpm). Taken together, these data demonstrate that MSCs, next to the anti-proliferative effect on T cells, have a profound inhibitory effect on the generation and function of both monocyte- and CD34+-derived DCs, indicating that MSCs are able to modulate immune responses at multiple levels.

Umbilical cord blood (UCB) is considered as an attractive alternative source of hematopoietic stem cells for allogeneic stem cell transplantations in patients who lack HLA-matched donors. However, the low cell dose adversely affects the speed of hematopoietic recovery and therefore limits the application of UCB transplantation in adults. Although ex-vivo expansion of cord blood cells has been explored as a strategy to increase the cell dose, compromised engraftment potential of expanded cells has been demonstrated. Another approach to overcome cell dose limitations is transplantation of multiple, unrelated UCB units. To investigate the effect of multiple cord transplantation on engraftment, NOD/SCID mice were transplanted with human hematopoietic progenitor cells (CD34+) derived from two UCB units with HLA disparity. During the first six weeks after transplantation the number of human platelets in peripheral blood was quantified by flow cytometry. Six weeks after transplantation, the mice were sacrificed and the percentage and donor origin of human CD45+ cells in blood, and in bone marrow was determined by flow cytometry. Transplantation of CD34+ cells derived from two UCB donors resulted in significantly higher number of human platelets in peripheral blood than transplantation of CD34+ cells from either donor alone, ranging from 3.92x106/ml to 10.29x106/ml (mean 6.4x106 ± 2.55x106/ml) and 0.11x106/ml to 3.12.106/ml (mean 1.42x106 ± 1.17x106/ml), respectively. Furthermore, the overall human cell engraftment level in bone marrow after double cord blood transplantation ranged from 7.01% to 64.34% (mean 29.6 ± 21.5%) a nearly 7-fold increase compared to single cord blood transplantation ranging from 0.27% to 13.5% (mean 4.6 ± 3.8%) Although consistently higher engraftment levels were reached after double cord blood transplantation, two different patterns were observed: in 2 out of 4 experiments cells from one donor predominated the engraftment (ratio 3:1), while in two other experiments the two units contributed equally to BM engraftment. The mechanism underlying these effects are is not yet clear. It is not very likely that the single donor predominance results from an unequal amount of hematopoietic stem cells in the cord blood units because each cord blood showed comparable levels of engraftment as a single unit. Alternatively, the unequal engraftment may result from an immunological competition or a graft versus graft stimulatory effect between the cords during the engraftment process and further studies are required to determine if the contribution of both units is dependent on the degree of HLA matching between the two cords. Taken together, these results demonstrate that double cord blood transplantation may represent a means of achieving increased engraftment, making multiple cord blood transplantation a promising strategy to improve the outcome of UCB transplantation. Studies are underway to unravel the mechanisms underlying the enhanced engraftment.