Person:

Kleffel, Sonja

Objective: Interleukin-21 (IL-21) is a proinflammatory cytokine that has been shown to affect Treg/Teff balance. However, the mechanism by which IL-21 orchestrates alloimmune response and interplays with Tregs is still unclear. Research design and methods: The interplay between IL-21/IL-21R signaling, FoxP3 expression, and Treg survival and function was evaluated in vitro in immunologically relevant assays and in vivo in allogenic and autoimmune models of islet transplantation. Results: IL-21R expression decreases on T cells and B cells in vitro and increases in the graft in vivo, while IL-21 levels increase in vitro and in vivo during anti-CD3/anti-CD28 stimulation/allostimulation in the late phase of the alloimmune response. In vitro, IL-21/IL-21R signaling (by using rmIL-21 or genetically modified (CD4^+) T cells [IL-21 pOrf plasmid–treated or hIL-21-Tg mice]) enhances the T-cell response during anti-CD3/anti-CD28 stimulation/allostimulation, prevents Treg generation, inhibits Treg function, induces Treg apoptosis, and reduces FoxP3 and FoxP3-dependent gene transcripts without affecting FoxP3 methylation status. In vivo targeting of IL-21/IL-21R expands intragraft and peripheral Tregs, promotes Treg neogenesis, and regulates the antidonor immune response, whereas IL-21/IL-21R signaling in Doxa-inducible ROSA-rtTA-IL-21-Tg mice expands Teffs and (FoxP3^−) cells. Treatment with a combination of mIL-21R.Fc and CTLA4-Ig (an inhibitor of the early alloimmune response) leads to robust graft tolerance in a purely alloimmune setting and prolonged islet graft survival in NOD mice. Conclusions: IL-21 interferes with different checkpoints of the FoxP3 Treg chain in the late phase of alloimmune response and, thus, acts as an antitolerogenic cytokine. Blockade of the IL-21/IL-21R pathway could be a precondition for tolerogenic protocols in transplantation.

Merkel cell carcinoma (MCC) is a highly virulent cutaneous neoplasm that, like melanoma, is a frequent cause of patient morbidity and mortality. The cellular mechanisms responsible for the aggressive behavior of MCC remain unknown. Vasculogenic mimicry (VM) is a phenomenon associated with cancer virulence, including in melanoma, whereby anastomosing laminin networks form in association with tumor cells that express certain endothelial genes. To determine whether VM is a factor in MCC, we employed a relevant xenograft model using two independent human MCC lines. Experimentally induced tumors were remarkably similar histologically to patient MCC, and both contained laminin networks associated with vascular endothelial-cadherin (CD144) and vascular endothelial growth factor receptor 1 (VEGFR-1) as well as Nodal expression typical of VM in melanoma. Moreover, two established chemotherapeutic agents utilized for human MCC, etoposide and carboplatin, induced necrosis in xenografts upon systemic administration while enriching for laminin networks in apparently resistant viable tumor regions that persisted. These findings for the first time establish VM-like laminin networks as a biomarker in MCC, demonstrate the experimental utility of the MCC xenograft model, and suggest that VM-rich regions of MCC may be refractory to conventional chemotherapeutic agents.

Matrix metalloproteinases (MMPs) are key biological mediators of processes as diverse as wound healing, embryogenesis, and cancer progression. Although MMPs may be induced through multiple signaling pathways, the precise mechanisms for their regulation in cancer are incompletely understood. Because cytoskeletal changes are known to accompany MMP expression, we sought to examine the potential role of the poorly understood cytoskeletal protein, nestin, in modulating melanoma MMPs. Nestin knockdown (KD) upregulated expression of specific MMPs and MMP-dependent invasion both through extracellular matrix barriers in vitro and in peritumoral connective tissue of xenografts in vivo. Development of 3-dimensionsal melanospheres that in vitro partially recapitulate non-invasive tumorigenic melanoma growth was inhibited by nestin KD, although ECM invasion by aberrant melanospheres that did form was enhanced. Mechanistically, nestin KD-dependent melanoma invasion was associated with intracellular redistribution of phosphorylated focal adhesion kinase (pFAK) and increased melanoma cell responsiveness to transforming growth factor-beta (TGF-β), both implicated in pathways of melanoma invasion. The results suggest that the heretofore poorly understood intermediate filament, nestin, may serve as a novel mediator of MMPs critical to melanoma virulence.