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Simple Summary SUMO is a ubiquitin-like post-translational modification important for many cellular processes and is suggested to play a role in cancer cell cycle progression. The aim of our study is to understand the role of SUMOylation in tumor progression and aggressiveness. Chondrosarcoma of bone was employed as a model to investigate if SUMOylation contributes to its aggressiveness. We confirmed that SUMO expression levels correlate with aggressiveness of chondrosarcoma and disease outcome. Inhibition of SUMOylation showed promising effects on reduction of chondrosarcoma growth in vitro. Our study implies that SUMO expression could be used as a potential biomarker for disease outcome in chondrosarcoma. Abstract Multiple components of the SUMOylation machinery are deregulated in various cancers and could represent potential therapeutic targets. Understanding the role of SUMOylation in tumor progression and aggressiveness would increase our insight in the role of SUMO in cancer and clarify its potential as a therapeutic target. Here we investigate SUMO in relation to conventional chondrosarcomas, which are malignant cartilage forming tumors of the bone. Aggressiveness of chondrosarcoma increases with increasing histological grade, and a multistep progression model is assumed. High-grade chondrosarcomas have acquired an increased number of genetic alterations. Using immunohistochemistry on tissue microarrays (TMA) containing 137 chondrosarcomas, we showed that higher expression of SUMO1 and SUMO2/3 correlates with increased histological grade. In addition, high SUMO2/3 expression was associated with decreased overall survival chances (p = 0. 0312) in chondrosarcoma patients as determined by log-rank analysis and Cox regression. Various chondrosarcoma cell lines (n = 7), especially those derived from dedifferentiated chondrosarcoma, were sensitive to SUMO inhibition in vitro. Mechanistically, we found that SUMO E1 inhibition interferes with cell division and as a consequence DNA bridges are frequently formed between daughter cells. In conclusion, SUMO expression could potentially serve as a prognostic biomarker.

For osteosarcoma (OS), the most common primary malignant bone tumor, overall survival has hardly improved over the last four decades. Especially for metastatic OS, novel therapeutic targets are urgently needed. A hallmark of cancer is aberrant metabolism, which justifies targeting metabolic pathways as a promising therapeutic strategy. One of these metabolic pathways, the NAD+ synthesis pathway, can be considered as a potential target for OS treatment. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the classical salvage pathway for NAD+ synthesis, and NAMPT is overexpressed in OS. In this study, five OS cell lines were treated with the NAMPT inhibitor FK866, which was shown to decrease nuclei count in a 2D in vitro model without inducing caspase-driven apoptosis. The reduction in cell viability by FK866 was confirmed in a 3D model of OS cell lines (n = 3). Interestingly, only OS cells with low nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1) RNA expression were sensitive to NAMPT inhibition. Using a publicly available (Therapeutically Applicable Research to Generate Effective Treatments (TARGET)) and a previously published dataset, it was shown that in OS cell lines and primary tumors, low NAPRT1 RNA expression correlated with NAPRT1 methylation around the transcription start site. These results suggest that targeting NAMPT in osteosarcoma could be considered as a novel therapeutic strategy, where low NAPRT expression can serve as a biomarker for the selection of eligible patients.

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.

Simple Summary Cartilage tumors frequently harbor mutations in the isocitrate dehydrogenase (IDH1 or IDH2) genes. These mutations cause an increase in the levels of the oncometabolite D-2-hydroxyglutarate (D-2-HG), which leads to widespread changes in several cellular processes, including the epigenetic landscape. The aim of our study was to explore whether the methylome of IDH mutant cartilage tumors is altered upon tumor progression and whether these underlying epigenetic vulnerabilities could be used as a target for therapy in both IDH wildtype and IDH mutant high-grade chondrosarcomas. As surgery is nowadays the only treatment option for chondrosarcoma patients, the identification of novel therapeutic strategies remains an important endeavor. The findings in this study show that histone deacetylase (HDAC) inhibition may represent a promising therapeutic strategy for all chondrosarcoma patients, since sensitivity towards this therapeutic option seems independent of the IDH mutation status and the chondrosarcoma subtype. Abstract Mutations in the isocitrate dehydrogenase (IDH1 or IDH2) genes are common in enchondromas and chondrosarcomas, and lead to elevated levels of the oncometabolite D-2-hydroxyglutarate causing widespread changes in the epigenetic landscape of these tumors. With the use of a DNA methylation array, we explored whether the methylome is altered upon progression from IDH mutant enchondroma towards high-grade chondrosarcoma. High-grade tumors show an overall increase in the number of highly methylated genes, indicating that remodeling of the methylome is associated with tumor progression. Therefore, an epigenetics compound screen was performed in five chondrosarcoma cell lines to therapeutically explore these underlying epigenetic vulnerabilities. Chondrosarcomas demonstrated high sensitivity to histone deacetylase (HDAC) inhibition in both 2D and 3D in vitro models, independent of the IDH mutation status or the chondrosarcoma subtype. siRNA knockdown and RNA expression data showed that chondrosarcomas rely on the expression of multiple HDACs, especially class I subtypes. Furthermore, class I HDAC inhibition sensitized chondrosarcoma to glutaminolysis and Bcl-2 family member inhibitors, suggesting that HDACs define the metabolic state and apoptotic threshold in chondrosarcoma. Taken together, HDAC inhibition may represent a promising targeted therapeutic strategy for chondrosarcoma patients, either as monotherapy or as part of combination treatment regimens.

Chondrosarcomas are chemo- and radiotherapy resistant and frequently harbor mutations in isocitrate dehydrogenase (IDH1 or IDH2), causing increased levels of D-2-hydroxyglutarate (D-2-HG). DNA repair defects and synthetic lethality with poly(ADP-ribose) polymerase (PARP) inhibition occur in IDH mutant glioma and leukemia models. Here we evaluated DNA repair and PARP inhibition, alone or combined with chemo- or radiotherapy, in chondrosarcoma cell lines with or without endogenous IDH mutations. Chondrosarcoma cell lines treated with the PARP inhibitor talazoparib were examined for dose&ndash, response relationships, as well as underlying cell death mechanisms and DNA repair functionality. Talazoparib was combined with chemo- or radiotherapy to evaluate potential synergy. Cell lines treated long term with an inhibitor normalizing D-2-HG levels were investigated for synthetic lethality with talazoparib. We report that talazoparib sensitivity was variable and irrespective of IDH mutation status. All cell lines expressed Ataxia Telangiectasia Mutated (ATM), but a subset was impaired in poly(ADP-ribosyl)ation (PARylation) capacity, homologous recombination, and O-6-methylguanine-DNA methyltransferase (MGMT) expression. Talazoparib synergized with temozolomide or radiation, independent of IDH1 mutant inhibition. This study suggests that talazoparib combined with temozolomide or radiation are promising therapeutic strategies for chondrosarcoma, irrespective of IDH mutation status. A subset of chondrosarcomas may be deficient in nonclassical DNA repair pathways, suggesting that PARP inhibitor sensitivity is multifactorial in chondrosarcoma.

Chondrosarcomas are chemo- and radiotherapy resistant and frequently harbor mutations in isocitrate dehydrogenase (IDH1 or IDH2), causing increased levels of D-2-hydroxyglutarate (D-2-HG). DNA repair defects and synthetic lethality with poly(ADP-ribose) polymerase (PARP) inhibition occur in IDH mutant glioma and leukemia models. Here we evaluated DNA repair and PARP inhibition, alone or combined with chemo- or radiotherapy, in chondrosarcoma cell lines with or without endogenous IDH mutations. Chondrosarcoma cell lines treated with the PARP inhibitor talazoparib were examined for dose–response relationships, as well as underlying cell death mechanisms and DNA repair functionality. Talazoparib was combined with chemo- or radiotherapy to evaluate potential synergy. Cell lines treated long term with an inhibitor normalizing D-2-HG levels were investigated for synthetic lethality with talazoparib. We report that talazoparib sensitivity was variable and irrespective of IDH mutation status. All cell lines expressed Ataxia Telangiectasia Mutated (ATM), but a subset was impaired in poly(ADP-ribosyl)ation (PARylation) capacity, homologous recombination, and O-6-methylguanine-DNA methyltransferase (MGMT) expression. Talazoparib synergized with temozolomide or radiation, independent of IDH1 mutant inhibition. This study suggests that talazoparib combined with temozolomide or radiation are promising therapeutic strategies for chondrosarcoma, irrespective of IDH mutation status. A subset of chondrosarcomas may be deficient in nonclassical DNA repair pathways, suggesting that PARP inhibitor sensitivity is multifactorial in chondrosarcoma.

Myxoid liposarcoma (MLS) is a soft tissue sarcoma characterized by a recurrent t(12;16) translocation. Although tumors are initially radio- and chemosensitive, the management of inoperable or metastatic MLS can be challenging. Therefore, our aim was to identify novel targets for systemic therapy. We performed an in vitro high-throughput drug screen using three MLS cell lines (402091, 1765092, DL-221), which were treated with 273 different drugs at four different concentrations. Cell lines and tissue microarrays were used for validation. As expected, all cell lines revealed a strong growth inhibition to conventional chemotherapeutic agents, such as anthracyclines and taxanes. A good response was observed to compounds interfering with Src and the mTOR pathway, which are known to be affected in these tumors. Moreover, BIRC5 was important for MLS survival because a strong inhibitory effect was seen at low concentration using the survivin inhibitor YM155, and siRNA for BIRC5 decreased cell viability. Immunohistochemistry revealed abundant expression of survivin restricted to the nucleus in all 32 tested primary tumor specimens. Inhibition of survivin in 402-91 and 1765-92 by YM155 increased the percentage S-phase but did not induce apoptosis, which warrants further investigation before application in the treatment of metastatic MLS. Thus, using a 273-compound drug screen, we confirmed previously identified targets (mTOR, Src) in MLS and demonstrate survivin as essential for MLS survival.

Background Conventional chondrosarcomas are malignant cartilage tumors considered radioresistant. Nevertheless, retrospective series show a small but significant survival benefit for patients with locally advanced disease treated with radiotherapy. And, in daily practice when considered inoperable their irradiation is an accepted indication for proton beam radiotherapy. Therefore, we investigated the sensitivity of chondrosarcoma cell lines and -tissue samples towards radiotherapy and screened for biomarkers to identify predictors of radiosensitivity. Methods Proliferation and clonogenic assays were performed in chondrosarcoma cell lines after γ-radiation in combination with mutant IDH1 inhibitor AGI-5198. In addition, glutathione levels were measured using mass spectrometry. Chondrosarcoma tumor explants were irradiated after which γ-H2AX foci were counted. Mutation analysis was performed using the Ion AmpliSeq™ Cancer Hotspot Panel and immunohistochemical staining’s were performed for P-S6, LC-3B, P53, Bcl-2, Bcl-xl and Survivin. Results were correlated with the number of γ-H2AX foci. Results Chondrosarcoma cell lines were variably γ-radiation resistant. No difference in radiosensitivity, nor glutathione levels was observed after treatment with AGI-5198. Irradiated chondrosarcoma patient tissue presented a variable increase in γ-H2AX foci compared to non-radiated tissue. Samples were divided into two groups, high and low radioresistant, based on the amount of γ-H2AX foci. All four highly resistant tumors exhibited mutations in the pRb pathway, while none of the less radioresistant tumors showed mutations in these genes. Conclusions Chondrosarcoma cell lines as well as primary tumors are variably radioresistant, particularly in case of a defective Rb pathway. Whether selection for radiotherapy can be based upon an intact Rb pathway should be further investigated. Electronic supplementary material The online version of this article (10.1186/s13569-019-0119-0) contains supplementary material, which is available to authorized users.

Background: Chondrosarcomas are malignant cartilage-producing tumors showing mutations and changes in gene expression in metabolism related genes. In this study, we aimed to explore the metabolome and identify targetable metabolic vulnerabilities in chondrosarcoma. Methods: A custom-designed metabolic compound screen containing 39 compounds targeting different metabolic pathways was performed in chondrosarcoma cell lines JJ012, SW1353 and CH2879. Based on the anti-proliferative activity, six compounds were selected for validation using real-time metabolic profiling. Two selected compounds (rapamycin and sapanisertib) were further explored for their effect on viability, apoptosis and metabolic dependency, in normoxia and hypoxia. In vivo efficacy of sapanisertib was tested in a chondrosarcoma orthotopic xenograft mouse model. Results: Inhibitors of glutamine, glutathione, NAD synthesis and mTOR were effective in chondrosarcoma cells. Of the six compounds that were validated on the metabolic level, mTOR inhibitors rapamycin and sapanisertib showed the most consistent decrease in oxidative and glycolytic parameters. Chondrosarcoma cells were sensitive to mTORC1 inhibition using rapamycin. Inhibition of mTORC1 and mTORC2 using sapanisertib resulted in a dose-dependent decrease in viability in all chondrosarcoma cell lines. In addition, induction of apoptosis was observed in CH2879 after 24 h. Treatment of chondrosarcoma xenografts with sapanisertib slowed down tumor growth compared to control mice. Conclusions: mTOR inhibition leads to a reduction of oxidative and glycolytic metabolism and decreased proliferation in chondrosarcoma cell lines. Although further research is needed, these findings suggest that mTOR inhibition might be a potential therapeutic option for patients with chondrosarcoma. Keywords: Chondrosarcoma, Metabolism, mTOR, Sapanisertib, Rapamycin

Introduction: Chondrosarcoma is a malignant cartilage-forming bone tumour in which mutations in IDH1 and IDH2 frequently occur. Previous studies suggest an increased dependency on glutaminolysis in IDH1/2 mutant cells, which resulted in clinical trials with the drugs CB-839, metformin and chloroquine. In this study, the preclinical rationale for using these drugs as a treatment for chondrosarcoma was evaluated. Methods: Expression of glutaminase was determined in 120 cartilage tumours by immunohistochemistry. Ten chondrosarcoma cell lines were treated with the metabolic compounds CB-849, metformin, phenformin (lipophilic analogue of metformin) and chloroquine. Results: A difference in glutaminase expression levels between the different tumour grades (p = 0.001, one-way ANOVA) was identified, with the highest expression observed in high-grade chondrosarcomas. Treatment with CB-839, metformin, phenformin or chloroquine revealed that chondrosarcoma cell lines are sensitive to glutaminolysis inhibition. Metformin and phenformin decreased mTOR activity in chondrosarcoma cells, and metformin decreased LC3B-II levels, which is counteracted by chloroquine. Conclusion: Targeting glutaminolysis with CB-839, metformin, phenformin or chloroquine is a potential therapeutic strategy for a subset of high-grade chondrosarcomas, irrespective of the presence or absence of an IDH1/2 mutation.

Background Ewing sarcoma is an aggressive, highly metastatic primary bone and soft tissue tumor most frequently occurring in the bone of young adolescents. Patients, especially those diagnosed with a metastatic disease, have a poor overall survival. Chemokine receptor CXCR4 has a key pro-tumorigenic role in the tumor microenvironment of Ewing sarcoma and has been suggested to be involved in the increased metastatic propensity. Earlier studies on CXCR4 protein expression in Ewing sarcoma yielded contradictory results when compared to CXCR4 RNA expression studies. Previously, we demonstrated that CXCR4 expression could be detected in vivo using the fluorescently tagged CXCR4-specific peptide MSAP-Ac-TZ14011. Therefore, we studied the membranous CXCR4 expression in Ewing sarcoma cell lines using MSAP-Ac-TZ14011. Methods The CXCR4 membrane expression levels were studied in EWS cell lines by flow cytometry using the hybrid peptide MSAP-Ac-TZ14011 and were correlated to CXCR4 RNA expression levels. The measurements were compared to levels detected using the CXCR4 antibody ab2074 under various cell preparation conditions. In addition, the staining patterns were analyzed by confocal fluorescence microscopy over time. Results The hybrid peptide MSAP-Ac-TZ14011 levels showed a strong and better correlation of CXCR4 membrane expression with the CXCR4 RNA expression levels than observed with the anti-CXCR4 antibody ab2074. With the hybrid peptide MSAP-Ac-TZ14011 using live cell confocal microscopy CXCR4 membrane staining and internalization was detected and the signal intensity correlated well with CXCR4 mRNA expression levels. Conclusions The fluorescently labeled CXCR4 targeting peptide-based method provides a reliable alternative to antibody staining to study the CXCR4 membrane expression in live cells using either flow cytometry or live cell fluorescence microscopy. The fluorescently tagged CXCR4 targeting peptide could enable in vivo detection of CXCR4 expression in Ewing sarcoma which may help to stratify cases for anti-CXCR4 therapy. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3352-z) contains supplementary material, which is available to authorized users.

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.

After allogeneic stem cell transplantation (allo-SCT), donor T cells may recognize minor histocompatibility antigens (MiHA) specifically expressed on cells of the recipient. It has been hypothesized that T cells recognizing hematopoiesis-restricted MiHA mediate specific graft-versus-leukemia (GVL) activity without inducing graft-versus-host disease (GVHD), whereas T cells recognizing ubiquitously expressed MiHA induce both GVL and GVHD reactivity. It also has been hypothesized that alloreactive CD4 T cells are capable of mediating specific GVL reactivity due to the hematopoiesis-restricted expression of HLA class II. However, clinical observations suggest that an overt GVL response, associated with expansion of T cells specific for hematopoiesis-restricted antigens, is often associated with GVHD reactivity. Therefore, we developed in vitro models to investigate whether alloreactive T cells recognizing hematopoiesis-restricted antigens induce collateral damage to surrounding nonhematopoietic tissues. We found that collateral damage to MiHA-negative fibroblasts was induced by misdirection of cytotoxic granules released from MiHA-specific T cells activated by MiHA-positive hematopoietic cells, resulting in granzyme-B–mediated activation of apoptosis in the surrounding fibroblasts. We demonstrated that direct contact between the activated T cell and the fibroblast is a prerequisite for this collateral damage to occur. Our data suggest that hematopoiesis-restricted T cells actively participate in an overt GVL response and may contribute to GVHD via induction of collateral damage to nonhematopoietic targets.

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.

Chondrosarcomas are malignant cartilage-forming bone tumors, which are intrinsically resistant to chemo- and radiotherapy, leaving surgical removal as the only curative treatment option. Therefore, our aim was to identify genes involved in chondrosarcoma cell survival that could serve as a target for therapy. siRNA screening for 51 apoptosis-related genes in JJ012 chondrosarcoma cells identified BIRC5, encoding survivin, as essential for chondrosarcoma survival. Using immunohistochemistry, nuclear as well as cytoplasmic survivin expression was analyzed in 207 chondrosarcomas of different subtypes. Nuclear survivin has been implicated in cell-cycle regulation while cytoplasmic localization is important for its anti-apoptotic function. RT–PCR was performed to determine expression of the most common survivin isoforms. Sensitivity to YM155, a survivin inhibitor currently in phase I/II clinical trial for other tumors, was examined in 10 chondrosarcoma cell lines using viability assay, apoptosis assay and cell-cycle analysis. Survivin expression was found in all chondrosarcoma patient samples. Higher expression of nuclear and cytoplasmic survivin was observed with increasing histological grade in central chondrosarcomas. Inhibition of survivin using YM155 showed that especially TP53 mutant cell lines were sensitive, but no caspase 3/7 or PARP cleavage was observed. Rather, YM155 treatment resulted in a block in S phase in two out of three chondrosarcoma cell lines, indicating that survivin is more involved in cell-cycle regulation than in apoptosis. Thus, survivin is important for chondrosarcoma survival and chondrosarcoma patients might benefit from survivin inhibition using YM155, for which TP53 mutational status can serve as a predictive biomarker.

Based on clinical observations that donor T cells specific for minor histocompatibility antigens (MiHA) ubiquitously expressed on both hematopoietic and nonhematopoietic cells were detected in patients showing evident graft-versus-leukemia/lymphoma (GVL) reactivity with no or limited coinciding graft-versus-host disease (GVHD), we hypothesized that nonhematopoietic tissues may be relatively unsusceptible to the cytotoxic effect of MiHA-specific T cells under normal, noninflammatory conditions. To test this hypothesis, we investigated the reactivity of alloreactive T cells specific for ubiquitously expressed MiHA against skin-derived primary human fibroblasts. We demonstrated that this reactivity was not merely determined by their antigen-specificity, but was highly dependent on adhesion molecule expression. ICAM-1 expression on the fibroblasts upregulated under proinflammatory conditions and induced during cross-talk with the T cells was demonstrated to be a crucial factor facilitating formation of high avidity interactions with the T cells and subsequent efficient target cell destruction. Furthermore, we provide supporting evidence for the role of ICAM-1 in vivo by demonstrating that ICAM-1 expression on nonhematopoietic target cells was dependent on the presence of infiltrating activated T cells, as was illustrated by restricted ICAM-1 expression at the sites of T cell infiltration in skin biopsies of patients with acute GVHD (aGVHD), by the absence of ICAM-1 expression in the same biopsies in areas without T cell infiltration and by the absence of ICAM-1 expression in biopsies of patients without GVHD independent of the presence of infiltrating nonactivated T cells. In conclusion, under noninflammatory conditions, nonhematopoietic tissues are unsusceptible to the GVHD reactivity of alloreactive T cells due to their inability to establish high avidity interactions.

After HLA class I-mismatched stem cell transplantation, allo-HLA–directed CD8 T cell responses can be activated without the help of CD4 T cells if memory CD8 T cells cross-reactive against the allo-HLA class I are present or if naive CD8 T cells are administered during inflammatory conditions. However, in the absence of inflammatory conditions, cooperation between CD4 and CD8 T cells likely is required for an effective primary CD8 T cell response directed against allo-HLA class I. In this study we investigated whether a coordinated response of CD8 and CD4 T cells could be demonstrated in an HLA class I–directed immune response in a patient who developed severe graft-versus-host disease (GVHD) after the administration HLA-A2–mismatched donor lymphocyte infusion in the absence of inflammatory conditions. A previously administered donor lymphocyte infusion from the same donor did not lead to an immune response, excluding the presence of a substantial pool of CD8 T cells cross-reactive against HLA-A2 within the memory T cell compartment of the donor. Analysis of isolated donor CD8 and CD4 T cell clones activated during the GVHD revealed a polyclonal CD8 T cell response directed against the mismatched HLA-A2 and a polyclonal CD4 T cell response recognizing HLA-A2–derived peptides presented in HLA class II. In addition, leukemic blasts present at the time of the emergence of GVHD expressed HLA-A2 and HLA class II and could activate both the CD4 and CD8 alloreactive T cells. Our results demonstrate that the GVHD was mediated by a cooperative CD4 and CD8 response directed against the mismatched HLA-A2 and suggest that leukemic blasts possibly activated this CD8 and CD4 T cell response.

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.

We previously found that human fetal lung is a rich source of mesenchymal stem cells (MSC). Here we characterize and analyze the frequency and function of MSC in other second-trimester fetal tissues.Single cell suspensions of fetal bone marrow (BM), liver, lung, and spleen were made and analyzed by flow cytometry for the expression of CD90, CD105, CD166, SH3, SH4, HLA-ABC, HLA-DR, CD34 and CD45. We assessed the frequency of MSC by limiting dilution assay.The frequency of MSC in BM was significantly higher than in liver, lung, and spleen (p0.05). On primary non-expanded cells from fetal liver, lung and spleen the number of cells positive for mesenchymal markers was significantly higher within the CD34 positive population than within the CD34 negative population. The phenotype of the culture-expanded MSC was similar for all fetal tissues, i.e. CD90, CD105, CD166, SH3, SH4 and HLA-ABC positive and CD34, CD45 and HLA-DR negative. Culture-expanded cells from all tissues were able to differentiate along adipogenic and osteogenic pathways. However, adipogenic differentiation was less in MSC derived from spleen, and osteogenic differentiation was reduced in liver-derived MSC (p0.05).Our results indicate that culture-expanded MSC derived from second-trimester fetal tissues, although phenotypically similar, exhibit heterogeneity in differentiating potential. We speculate that these differences may be relevant for the clinical application of MSC.

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.