IntroductionHuman bone marrow stromal cells, also referred to as mesenchymal stem cells (MSCs), are able to differentiate along multiple lineages such as chondrocytes, osteoblasts, adipocytes, myocytes, and astrocytes. 1 MSCs, rare residents in the bone marrow, can be rapidly expanded ex vivo without loss of their multilineage differentiation potential. Because of their ability to migrate to sites of tissue injury, 2,3 MSCs have emerged as a promising therapeutic modality for tissue regeneration and repair. Several studies in animal models have demonstrated that MSCs are capable of long-term engraftment and in vivo differentiation, and encouraging results have been reported in clinical use. [4][5][6] MSCs are known to secrete a number of cytokines and regulatory molecules implicated in different aspects of hematopoiesis. 7 These characteristics have generated clinical interest to use MSCs to enhance hematopoietic stem cell engraftment. Although animal models provide experimental evidence that MSCs facilitate engraftment, 8,9 no conclusive evidence has yet been presented in humans. 6 In addition to providing critical growth factors, MSCs display immunosuppressive properties that might facilitate engraftment. In vitro studies with human, baboon, and murine MSCs demonstrated that MSCs suppress the proliferation of T cells induced by alloantigens or mitogens. 10-12 Furthermore, MSCs have been reported to induce T-cell division arrest, 13 to inhibit the differentiation and maturation of dendritic cells, 14,33 and to decrease the production of inflammatory cytokines by various immune cell populations. 15 Controversy exists regarding their effect on cytotoxic T cells and NK cells. 16,17 Animal studies indicate that, in line with their immunosuppressive capacities in vitro, MSCs also display immunosuppressive capacities in vivo; allogeneic MSCs may prolong skin allograft survival in immunocompetent baboons 10 and may prevent the rejection of allogeneic tumor cells in immunocompetent mice. 18 The mechanisms underlying these effects of MSCs have not been clearly identified. Although conflicting results have been reported, most studies agree that soluble factors are involved. 11,[18][19][20] The therapeutic application of the immunosuppressive properties of MSCs has already been exploited in the clinical setting for the treatment of acute graftversus-host disease after allogeneic stem cell transplantation. 21 The immunophenotype of MSCs, the low expression of human leukocyte antigen (HLA) major histocompatibility complex (MHC) class I, and the absence of costimulatory molecules, together with the observation that MSCs do not elicit a proliferative response from allogeneic lymphocytes, suggest that MSCs are of inherently low immunogenicity. 11,20 These properties might open attractive possibilities to use universal donor MSCs for different therapeutic applications.The aim of this study was to examine whether MSCs display immunosuppressive properties in vivo in murine allogeneic bone marrow transplantation models. The transp...

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.

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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.

Key Points• GVHD after HLA-DPB1-mismatched CD41 DLI after TCD-alloSCT is mediated by allo-reactive HLA-DPB1-directed CD41 T cells.• Viral infections after TCDalloSCT can induce HLA class II on nonhematopoietic tissues, making them targets for CD41 T cells in GVHD.CD81 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 CD41 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 CD41 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 CD41 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 CD41 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 CD41 T cells, which in turn could target nonhematopoietic tissues in GVHD. In conclusion, our data demonstrate that GVHD after HLA-DPB1-mismatched CD41 DLI can be mediated by allo-reactive HLA-DPB1-directed CD41 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. (Blood. 2013;122(11):1963-1973 IntroductionIn allogeneic hematopoietic stem cell transplantation (alloSCT), T-cell depletion of the graft efficiently prevents the occurrence of severe acute graft-versus-host disease [GVHD]) 1,2 but also adversely affects posttransplant antitumor and antipathogen immunity.1-3 Early intervention with donor lymphocyte infusion (DLI) after T cell-depleted (TCD)-alloSCT may prevent relapse of the malignancy and improve immune reconstitution against pathogens but is frequently associated with reintroduction of GVHD. 4,5 Therefore, exploration of treatment strategies to improve the balance between graft-versus-leukemia (GVL) reactivity and antipathogen immunity and GVHD is warranted.To minimize the risk of GVHD, patients are preferably transplanted with stem cell grafts from HLA-matched sibling or unrelated donors (URD).6 HLA matching is generally performed for HLA-A, -B, -C, -DRB1, and -DQB1 alleles (10/10 match) but not for HLA-DPB1. Therefore, 70% to 80% of URD alloSCT are HLA-DPB1-mismatched. 6,7 In contrast to HLA class I, constitutive expression of HLA class II molecules is mainly confined to normal...