The forkhead transcription factor Foxp3 is highly expressed in CD4+CD25+ regulatory T cells (Treg) and was recently identified as a key player in mediating their inhibitory functions. Here, we describe for the first time the expression and function of Foxp3 in pancreatic ductal adenocarcinoma cells and tumors. Foxp3 expression was induced by transforming growth factor-B2 (TGF-B2), but not TGF-B1 stimulation in these cells, and was partially suppressed following antibody-mediated neutralization of TGF-B2. The TGF-B2 effect could be mimicked by ectopic expression of a constitutively active TGF-B type I receptor/ALK5 mutant. Down-regulation of Foxp3 with small interfering RNA (siRNA) in pancreatic carcinoma cells resulted in the up-regulation of interleukin 6 (IL-6) and IL-8 expression, providing evidence for a negative transcriptional activity of Foxp3 also in these epithelial cells. Coculture of Foxp3-expressing tumor cells with naive T cells completely inhibited T-cell proliferation, but not activation, and this antiproliferative effect was partially abrogated following specific inhibition of Foxp3 expression. These findings indicate that pancreatic carcinoma cells share growth-suppressive effects with Treg and suggest that mimicking Treg function may represent a new mechanism of immune evasion in pancreatic cancer. [Cancer Res 2007;67(17):8344-50]

Regulatory macrophages (M regs) were administered to two living-donor renal transplant recipients. Both patients were minimized to low-dose tacrolimus monotherapy within 24 wk of transplantation and subsequently maintained excellent graft function. After central venous administration, most M regs remained viable and were seen to traffic from the pulmonary vasculature via the blood to liver, spleen, and bone marrow. By 1 y posttransplantation, both patients displayed patterns of peripheral blood gene expression converging upon the IOT-RISET signature. Furthermore, both patients maintained levels of peripheral blood FOXP3 and TOAG-1 mRNA expression within the range consistent with nonrejection. It is concluded that M regs warrant further study as a potential immune-conditioning therapy for use in solid-organ transplantation. The results of this work are being used to inform the design of The ONE Study, a multinational clinical trial of immunomodulatory cell therapy in renal transplantation.

The ability of human gd T cells from healthy donors to kill pancreatic ductal adenocarcinoma (PDAC) in vitro and in vivo in immunocompromised mice requires the addition of gd T-cell-stimulating antigens. In this study, we demonstrate that gd T cells isolated from patients with PDAC tumor infiltrates lyse pancreatic tumor cells after selective stimulation with phosphorylated antigens. We determined the absolute numbers of gd T-cell subsets in patient whole blood and applied a real-time cell analyzer to measure their cytotoxic effector function over prolonged time periods. Because phosphorylated antigens did not optimally enhance gd T-cell cytotoxicity, we designed bispecific antibodies that bind CD3 or Vg9 on gd T cells and Her2/neu (ERBB2) expressed by pancreatic tumor cells. Both antibodies enhanced gd T-cell cytotoxicity with the Her2/Vg9 antibody also selectively enhancing release of granzyme B and perforin. Supporting these observations, adoptive transfer of gd T cells with the Her2/Vg9 antibody reduced growth of pancreatic tumors grafted into SCID-Beige immunocompromised mice. Taken together, our results show how bispecific antibodies that selectively recruit gd T cells to tumor antigens expressed by cancer cells illustrate the tractable use of endogenous gd T cells for immunotherapy.

The interaction of the MHC class I-related chain molecules A and B (MICA and MICB) with the corresponding natural killer group 2, member D (NKG2D) receptor triggers cytotoxic effector activity of natural killer cells and certain T-cell subsets and provides a costimulatory signal for cytokine production. Thus, the presence of MICA=B on transformed cells contributes to tumor immunosurveillance. Consequently, the proteolytic cleavage of MICA=B is regarded as an important immune escape mechanism of various cancer cells. To investigate the molecular machinery responsible for the shedding of endogenous MICA=B, we analyzed different human tumor entities including mammary, pancreatic and prostate carcinomas. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) revealed that all tested tumor cells constitutively expressed MICA and MICB on the cell surface and also released NKG2D ligands into the supernatant. We demonstrate that the "a disintegrin and metalloproteases" (ADAMs) 10 and 17 are largely responsible for the generation of soluble MICA=B. Pharmacological inhibition of metalloproteases reduced the level of released MICA=B and increased cell surface expression. Studies using RNA interference not only revealed a prominent role of ADAM10 and ADAM17 in NKG2D ligand shedding but also a tumor cell-specific role of ADAM10 and=or ADAM17 in shedding of MICA or MICB. Moreover, we report that in the prostate carcinoma cell line PC-3, MICA was not shed at all but rather was secreted in exosomes. These data indicate that the release of NKG2D ligands from individual tumor entities is by far more complex than suggested in previously reported MICA=B transfection systems.The activating natural killer group 2, member D (NKG2D) receptor is expressed on NK cells, NKT cells, gd T cells, CD8 1 T cells and a minor immunoregulatory subset of CD4 1 T cells. 1,2 The ligation of NKG2D costimulates T cells but also directly triggers cell-mediated cytotoxicity and cytokine release in NK cells. [3][4][5][6] Thus, despite some exceptions including gastrointestinal epithelium 7 and placenta, 8 the ligands for NKG2D are not expressed on healthy tissues to avoid inadvertent damage. However, NKG2D ligand (NKG2DL) expression can be induced in response to a variety of stimuli related to cellular stress such as heat shock, viral infection, DNA damage, oxidative stress and certain proinflammatory signals. 9,10 Of note, many tumors and precancerous lesions express NKG2D ligands presumably as a bystander effect of the oncogenic process itself. 11 As NKG2DL expression renders tumors susceptible to NKG2D 1 lymphocytes, the NKG2D system plays a pivotal role in immunosurveillance. 12 On the other hand, the sustained exposure to tumor cell-bound NKG2D ligands might also result in surface modulation and functional impairment of the NKG2D receptor and induce cross-tolerance of additional NK cell activation pathways. 13,14 A hallmark of the NKG2D system is the multitude of ligands identified so far. In humans, these include the MHC class I-related chain ...

How distinct stem cell populations originate and whether there is a clear stem cell "genetic signature" remain poorly understood. Understanding the evolution of stem cells requires molecular profiling of stem cells in an animal at a basal phylogenetic position. In this study, using transgenic Hydra polyps, we reveal for each of the three stem cell populations a specific signature set of transcriptions factors and of genes playing key roles in cell type-specific function and interlineage communication. Our data show that principal functions of stem cell genes, such as maintenance of stemness and control of stem cell self-renewal and differentiation, arose very early in metazoan evolution. They are corroborating the view that stem cell types shared common, multifunctional ancestors, which achieved complexity through a stepwise segregation of function in daughter cells.

TLR3 recognizes viral dsRNA and its synthetic mimetic polyinosinic-polycytidylic acid (poly(I:C)). TLR3 expression is commonly considered to be restricted to dendritic cells, NK cells, and fibroblasts. In this study we report that human γδ and αβ T lymphocytes also express TLR3, as shown by quantitative real-time PCR, flow cytometry, and confocal microscopy. Although T cells did not respond directly to poly(I:C), we observed a dramatic increase in IFN-γ secretion and an up-regulation of CD69 when freshly isolated γδ T cells were stimulated via TCR in the presence of poly(I:C) without APC. IFN-γ secretion was partially inhibited by anti-TLR3 Abs. In contrast, poly(I:C) did not costimulate IFN-γ secretion by αβ T cells. These results indicate that TLR3 signaling is differentially regulated in TCR-stimulated γδ and αβ T cells, suggesting an early activation of γδ T cells in antiviral immunity.

Recent evidence suggests that natural selection operating on hosts to maintain their microbiome contributes to the emergence of new species, that is, the ‘hologenomic basis of speciation’. Here we analyse the gut microbiota of two house mice subspecies, Mus musculus musculus and M. m. domesticus, across their Central European hybrid zone, in addition to hybrids generated in the lab. Hybrid mice display widespread transgressive phenotypes (that is, exceed or fall short of parental values) in a variety of measures of bacterial community structure, which reveals the importance of stabilizing selection operating on the intestinal microbiome within species. Further genetic and immunological analyses reveal genetic incompatibilities, aberrant immune gene expression and increased intestinal pathology associated with altered community structure among hybrids. These results provide unique insight into the consequences of evolutionary divergence in a vertebrate ‘hologenome’, which may be an unrecognized contributing factor to reproductive isolation in this taxonomic group.

Hydra ’s unlimited life span has long attracted attention from natural scientists. The reason for that phenomenon is the indefinite self-renewal capacity of its stem cells. The underlying molecular mechanisms have yet to be explored. Here, by comparing the transcriptomes of Hydra ’s stem cells followed by functional analysis using transgenic polyps, we identified the transcription factor forkhead box O (FoxO) as one of the critical drivers of this continuous self-renewal. foxO overexpression increased interstitial stem cell and progenitor cell proliferation and activated stem cell genes in terminally differentiated somatic cells. foxO down-regulation led to an increase in the number of terminally differentiated cells, resulting in a drastically reduced population growth rate. In addition, it caused down-regulation of stem cell genes and antimicrobial peptide (AMP) expression. These findings contribute to a molecular understanding of Hydra ’s immortality, indicate an evolutionarily conserved role of FoxO in controlling longevity from Hydra to humans, and have implications for understanding cellular aging.