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.

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.

Patients with malignant diseases can be effectively treated with allogeneic hematopoietic stem cell transplantation (allo-SCT). Polymorphic peptides presented in HLA molecules, the so-called minor histocompatibility antigens (MiHA), play a crucial role in antitumor immunity as targets for alloreactive donor T cells. Identification of multiple MiHAs is essential to understand and manipulate the development of clinical responses after allo-SCT. In this study, CD8+ T-cell clones were isolated from leukemia patients who entered complete remission after allo-SCT, and MiHA-specific T-cell clones were efficiently selected for analysis of recognition of a panel of EBV-transformed B cells positive for the HLA restriction elements of the selected T-cell clones. One million single nucleotide polymorphisms (SNP) were determined in the panel cell lines and investigated for matching with the T-cell recognition data by whole genome association scanning (WGAs). Significant association with 12 genomic regions was found, and detailed analysis of genes located within these genomic regions revealed SNP disparities encoding polymorphic peptides in 10 cases. Differential recognition of patient-type, but not donor-type, peptides validated the identification of these MiHAs. Using tetramers, distinct populations of MiHA-specific CD8 + T cells were detected, demonstrating that our WGAs strategy allows high-throughput discovery of relevant targets in antitumor immunity after allo-SCT.

The alloreactive human T cell clone MBM15 was found to exhibit dual specificity recognizing both an antigen in the context of the HLA class I A2 molecule and an antigen in the context of the HLA class II DR1. We demonstrated that the dual reactivity that was mediated via a single clonal T cell population depended on specific peptide binding. For complete recognition of the HLA-A2-restricted specificity the interaction of CD8 with HLA class I is essential. Interestingly, interaction of the CD8 molecule with HLA class I contributed to the HLA-DR1-restricted specificity. T cell clone MBM15 expressed two in-frame T cell receptor (TCR) V␣ transcripts (V␣1 and V␣2) and one TCR V␤ transcript (V␤13). To elucidate whether two TCR complexes were responsible for the dual recognition or one complex, cytotoxic T cells were transduced with retroviral vectors encoding the different TCR chains. Only T cells transduced with the TCR V␣1V␤13 combination specifically recognized both the HLA-A2 ؉ and HLA-DR1 ؉ target cells, whereas the V␣2V␤13 combination did not result in a TCR on the cell surface. T he function of the major histocompatibility complex (MHC) molecules is to bind and display peptides to T lymphocytes. Allogeneic MHC molecules can induce strong T cell responses, which is reflected by the mixed lymphocyte reaction in vitro and the high incidence of graft rejection and graft versus host disease after transplantation of organs or hematopoietic cells over MHC barriers. T cell recognition of allogeneic MHC is often peptide specific, resembling self-MHC-restricted T cell recognition of foreign antigens (1-3). However, alloreactive T cells are heterogeneous in their degree of peptide specificity (4), and a minor population might be peptide independent or recognize motifs shared by many peptides (5). Several alloreactive T cell clones have been described to be crossreactive, recognizing two unrelated peptides in the context of two different allogeneic MHC class I molecules (6, 7). In addition, alloreactive cytotoxic T lymphocytes recognizing an endogenously processed peptide binding to allogeneic MHC molecules and recognizing a different peptide in the context of self-MHC class I have been described (8). In one instance, crossreactive cytotoxic T lymphocytes showing dual recognition for both HLA class I and class II molecules also have been reported (9, 10). Those authors postulated that based on the shared structural motif between the HLA-B27 and the DR2 B5*0101 chain, the reactivity pattern reflected presentation of identical or structurally related peptides by HLA-B27 and HLA-DR2. In several of these previously mentioned studies, cold-target inhibition experiments were performed to confirm that one clonal T cell population was mediating the crossreactivity. However, whether the crossreactivity of these alloreactive T cell clones was mediated via one or two T cell receptor (TCR) ␣␤ complexes was not investigated. This hypothesis may be possible because 20% of peripheral human T cells and 10% of mouse T cells express two dif...