Human mesenchymal stem cells (hMSC) aid in tissue maintenance and repair by differentiating into specialized cell types. Due to this ability, hMSC are currently being evaluated for cell-based therapies of tissue injury and degenerative diseases. However, extensive expansion ex vivo is a prerequisite to obtain the cell numbers required for human cell-based therapy protocols. Recent studies indicate that hMSC may contribute to cancer development and progression either by acting as cancer-initiating cells or through interactions with stromal elements. If spontaneous transformation ex vivo occurs, this may jeopardize the use of hMSC as therapeutic tools. Whereas murine MSC readily undergo spontaneous transformation, there are conflicting reports about spontaneous transformation of hMSC. We have addressed this controversy in a two-center study by growing bone marrow-derived hMSC in long-term cultures (5-106 weeks). We report for the first time spontaneous malignant transformation to occur in 45.8% (11 of 24) of these cultures. In comparison with hMSC, the transformed mesenchymal cells (TMC) showed a significantly increased proliferation rate and altered morphology and phenotype. In contrast to hMSC, TMC grew well in soft agar assays and were unable to undergo complete differentiation. Importantly, TMC were highly tumorigenic, causing multiple fastgrowing lung deposits when injected into immunodeficient mice. We conclude that spontaneous malignant transformation may represent a biohazard in long-term ex vivo expansion of hMSC. On the other hand, this spontaneous transformation process may represent a unique model for studying molecular pathways initiating malignant transformation of hMSC. [Cancer Res 2009;69(13):5331-9]

It is generally accepted that priming of antitumor CD8 ؉ cytotoxic T lymphocytes (CTLs) needs help that can be provided by CD4 ؉ T cells. We show that interactions between dendritic cells (DCs) and natural killer (NK) cells can bypass the T helper arm in CTL induction. Bone marrow-derived DCs caused rejection of the A20 lymphoma and induced tumor-specific long-term memory, although they were not loaded with tumor-derived antigen. Experiments using CD40 ؊ knock-out mice and cell depletion showed that this effect did not require CD4 ؉ cells. Both primary rejection and long-term CTL memory were the result of NK cell activation by DCs. NK cytotoxicity, which was necessary for primary rejection, was dependent on expression of natural killer group 2 D (NKG2D) ligands on tumor cells. Blocking of these ligands using NKG2D tetramers abrogated tumor killing in vitro and in vivo. The long-term response was due to CTLs directed against antigen(s) expressed on A20 and in vitrodifferentiated DCs. The mechanism leading to CD4 ؉ helper cell-independent CTL responses was elucidated as a cascade that was initiated by NK cell activation. This pathway was dependent on interferon-␥ expression and involved priming endogenous DCs for interleukin-12 production. Our data suggest a novel pathway linking innate and adaptive immunity. ( IntroductionInduction of efficient immune responses requires a coordinated interplay between innate and adaptive immune effector systems. Dendritic cells (DCs) are components of the innate immune system that activate specific effectors of adaptive immunity. 1,2 In an immature state, DCs are able to ingest antigen (Ag). Following a maturation process that involves migration to lymphoid tissues, down-regulation of Ag uptake and upregulation of major histocompatibility complex (MHC) and costimulatory molecules, DCs present antigenic peptides to T lymphocytes. 1 Exogenous proteins are taken up and processed by DCs and presented to CD4 ϩ cells in association with MHC class II molecules, whereas intracellular Ags are presented by MHC class I molecules to CD8 ϩ cytotoxic T lymphocytes (CTLs). There is, however, emerging evidence that exogenous proteins can also be directed to the endogenous presentation pathway, thus leading to CTL induction, a process referred to as cross-presentation. Efficient generation of CTLs from naive CD8 ϩ T cells needs help from CD4 ϩ T cells. [3][4][5] This help involves secretion of cytokines and CD40/CD40L interactions that lead to increased expression of costimulatory molecules on DCs and to induction of interleukin-12 (IL-12). 6 As shown in mouse models, CD4 ϩ T cells are pivotal for protection against tumors and can even mediate tumor rejection independently of CD8 ϩ T lymphocytes, if they are biased toward a T helper 1 (Th1) response. 7 Expression of CD40 by DCs is crucial for the production of Th1 cytokines such as IL-12 and for tumor protection. 7,8 Natural killer (NK) cells are effector cells of the innate immune system that exert direct cytotoxic functions. 9 These are determ...

No abstract

Bispecific antibodies (bsAb) are considered as promising tools for the elimination of disseminated tumour cells in a minimal residual disease situation. The bsAb-mediated recruitment of an immune effector cell in close vicinity of a tumour cell is thought to induce an antitumoural immune response. However, classical bispecific molecules activate only a single class of immune effector cell that may not yield optimal immune responses. We therefore constructed an intact bispecific antibody, BiUII (anti-CD3 × anti-EpCAM), that not only recognizes tumour cells and T lymphocytes with its two binding arms, but also binds and activates Fcγ-receptor positive accessory cells through its Fc-region. We have demonstrated recently that activated accessory cells contribute to the bsAb-induced antitumoural activity. We now analyse this stimulation in more detail and demonstrate here the BiUll-induced upregulation of activation markers like CD83 and CD95 on accessory cells and the induction of neopterin and biopterin synthesis. Experiments with pure cell subpopulations revealed binding of BiUll to CD64+ accessory cells and CD16+ NK cells, but not to CD32+ B lymphocytes. We provide further evidence for the importance of the Fc-region in that this bispecific molecule stimulates Fcγ-R-positive accessory cells to eliminate tumour cells in vitro by direct phagocytosis. © 2000 Cancer Research Campaign