Natural killer (NK) cells are the first lymphocytes to recover after allogeneic stem cell transplantation (SCT) and can exert powerful graft-versus-leukemia (GVL) effects determining transplant outcome. Conditions governing NK cell alloreactivity and the role of NK recovery in sibling SCT are not well defined. NK cells on day 30 post-transplant (NK30) were measured in 54 SCT recipients with leukemia and donor and recipient killer immunoglobulin-like receptor (KIR) genotype determined. In univariate analysis, donor KIR genes 2DL5A, 2DS1, 3DS1 (positive in 46%) and higher numbers of inhibitory donor KIR correlated with higher NK30 counts and were associated with improved transplant outcome. NK30 counts also correlated directly with the transplant CD34 cell dose and inversely with the CD3 þ cell dose. In multivariate analysis, the NK30 emerged as the single independent determinant of transplant outcome. Patients with NK30 4150/ll had less relapse (HR 18.3, P ¼ 0.039), acute graft-versus-host disease (HR 3.2, P ¼ 0.03), non-relapse mortality (HR 10.7, P ¼ 0.028) and improved survival (HR 11.4, P ¼ 0.03). Results suggest that T cell-depleted SCT might be improved and the GVL effect enhanced by selecting donors with favorable KIR genotype, and by optimizing CD34 and CD3 doses.

IntroductionNatural killer (NK) cells have antigen-independent tumor cytotoxicity and have been shown in murine models to control and prevent tumor growth and dissemination. 1,2 However, the exact role that NK cells play in the control of cancer in humans remains a matter of controversy.NK cells do not rearrange genes coding for specific antigen receptors; rather, their recognition of targets is regulated through a balance of activating or inhibitory signals. 3 It is necessary to inactivate NK cells to prevent their destruction of normal host tissues. 4 Therefore, even in the presence of an activating ligand, inhibitory ligands expressed on cells may deliver overriding signals that culminate in a net suppression of NK cell function. Recently, a growing number of NK cell inhibitory and activating receptors were characterized. 5-8 NK cells can recognize major histocompatibility (MHC) class I and class I-like molecules through killer immunoglobulin-like receptors (KIRs) expressed on their surfaces. MHC class I ligation of KIRs on normal and malignant tissues suppresses NK cell function. MHC class I molecules fall into groups that serve as ligands for specific KIR, resulting in the inhibition of NK cell-mediated cytotoxicity. Polymorphisms in amino acids residing at positions 77 and 80 of HLA-C dictate specificity for its target KIR. 9-11 KIR2DL1 recognizes group 2 HLA-C molecules that have an asparagine at position 77 and a lysine at position 80, whereas KIR2DL2 and KIR2DL3 recognize group 1 HLA-C molecules that have a serine at position 77 and an asparagine at position 80.The inactivation of NK cells by self-HLA molecules might be a mechanism permitting malignant host cells to evade NK cellmediated immunity. Because tumor KIR ligands are always matched to NK cell KIR, autologous NK cells would be inhibited by MHC class I-expressing tumors, even in the presence of activating ligands. This may in part explain the failure of adoptively transfused autologous NK 12 or lymphokine-activated killer (LAK) cells to mediate antitumor effects against most metastatic solid tumors. 13 Only tumor cells that have lost MHC class I expression or have a dominant-activating ligand are predicted to be susceptible to such populations. 14 Ruggeri et al 15,16 and others 17 have shown allogeneic NK cells can mediate antileukemic effects against AML after allogeneic haploidentical and partially mismatched unrelated hematopoietic cell transplantation when KIR/KIR ligand incompatibility exists in the graft-versus-host (GVH) direction (defined as an MHC class I KIR ligand that is absent in the recipient but present in the donor). In this setting, donor NK cells expand that are not inhibited by For personal use only. on March 22, 2019. by guest www.bloodjournal.org From ligands expressed on recipient leukemia cells, substantially reducing the risk for disease relapse compared with those who receive KIR-compatible transplants. This beneficial effect does not appear to occur against acute lymphoblastic leukemia (ALL), perhaps because these popul...

Alternatively spliced tissue factor (asTF) promotes neovascularization and monocyte recruitment via integrin ligation. While asTF mRNA has been detected in some pancreatic ductal adenocarcinoma (PDAC) cell lines and increased asTF expression can promote PDAC growth in a subcutaneous model, the expression of asTF protein in bona fide PDAC lesions and/or its role in metastatic spread are yet to be ascertained. We here report that asTF protein is abundant in lesional and stromal compartments of the five studied types of carcinoma including PDAC. Analysis of 29 specimens of PDAC revealed detectable asTF in >90% of the lesions with a range of staining intensities. asTF levels in PDAC lesions positively correlated with the degree of monocyte infiltration. In an orthotopic model, asTF-overexpressing high-grade PDAC cell line Pt45P1/asTF+ produced metastases to distal lymph nodes, which stained positive for asTF. PDAC cells stimulated with and/or overexpressing asTF exhibited upregulation of genes implicated in PDAC progression and metastatic spread. Pt45P1/asTF+ cells displayed higher coagulant activity compared to Pt45P1 cells; the same effect was observed for cell-derived microparticles (MPs). Our findings demonstrate that asTF is expressed in PDAC and lymph node metastases and potentiates PDAC spread in vivo. asTF elicits global changes in gene expression likely involved in tumor progression and metastatic dissemination, and it also enhances the pro-coagulant potential of PDAC cells and cell-derived MPs. Thus, asTF may comprise a novel therapeutic target to treat PDAC and, possibly, its thrombotic complications.

Objective Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. Approach and Results We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet (HFD) to the carotid arteries of recipient HFD-fed LDLR−/− mice. Two weeks after transplantation, wire injury was performed, and animals were sacrificed two weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization, and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed α-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of MCP-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. Conclusions These findings indicate that PVAT contributes to the vascular response to wire injury, in part through MCP-1-dependent mechanisms.

Ang-2 mRNA and protein increased in the retinal tissues after 8 weeks of diabetes and in high-glucose-treated cells. Intravitreal injection of Ang-2 in rats produced a significant increase in retinal vascular permeability. Ang-2 increased HREC monolayer permeability that was associated with a decrease in VE-cadherin and a change in monolayer morphology. High glucose and Ang-2 produced a significant increase in VE-cadherin phosphorylation. CONCLUSIONS; Ang-2 is upregulated in the retina in an animal model of diabetes, and hyperglycemia induces the expression of Ang-2 in isolated retinal endothelial cells. Increased Ang-2 alters VE-cadherin function, leading to increased vascular permeability. Thus, Ang-2 may play an important role in increased vasopermeability in diabetic retinopathy.

Rationale: Hemizygous deficiency of the transcription factor Krüppel-like factor 2 (KLF2) has been shown previously to augment atherosclerosis in hypercholesterolemic mice. However, the cell type responsible for the increased atherosclerosis due to KLF2 deficiency has not been identified. This study examined the consequence of myeloid cell-specific KLF2 inactivation in atherosclerosis. Methods and Results: Cell-specific knockout mice were generated by Cre/ loxP recombination. Macrophages isolated from myeloid-specific Klf2 knockout ( myeKlf2 −/− ) mice were similar to myeKlf2 +/+ macrophages in response to activation, polarization, and lipid accumulation. However, in comparison to myeKlf2 +/+ macrophages, myeKlf2 −/− macrophages adhered more robustly to endothelial cells. Neutrophils from myeKlf2 −/− mice also adhered more robustly to endothelial cells, and fewer myeKlf2 −/− neutrophils survived in culture over a 24-hour period in comparison with myeKlf2 +/+ neutrophils. When myeKlf2 −/− mice were mated to Ldlr −/− mice and then fed a high fat and high cholesterol diet, significant increase in atherosclerosis was observed in the myeKlf2 −/− Ldlr −/− mice compared with myeKlf2 +/+ Ldlr −/− littermates. The increased atherosclerosis in myeKlf2 −/− Ldlr −/− mice was associated with elevated presence of neutrophils and macrophages, with corresponding increase of myeloperoxidase as well as chlorinated and nitrosylated tyrosine epitopes in their lesion areas compared with myeKlf2 +/+ Ldlr −/− mice. Conclusions: This study documents a role for myeloid KLF2 expression in modulating atherosclerosis. The increased neutrophil accumulation and atherosclerosis progression with myeloid-specific KLF2 deficiency also underscores the importance of neutrophils in promoting vascular oxidative stress and atherosclerosis. Collectively, these results suggest that elevating KLF2 expression may be a novel strategy for prevention and treatment of atherosclerosis.

Summary Background TF is highly expressed in cancerous and atherosclerotic lesions. Monocyte recruitment is a hallmark of disease progression in these pathological states. Objective To examine the role of integrin signaling in TF-dependent recruitment of monocytes by endothelial cells. Methods The expression of flTF and asTF in cervical cancer and atherosclerotic lesions was examined. Biologic effects of the exposure of primary microvascular endothelial cells (MVEC) to truncated flTF ectodomain (LZ-TF) and recombinant asTF were assessed. Results flTF and asTF exhibited nearly identical expression patterns in cancer lesions and lipid-rich plaques. Tumor lesions as well as stromal CD68+ monocytes/macrophages expressed both TF forms. Primary MVEC rapidly adhered to asTF and LZ-TF, and this was completely blocked by anti-β1 integrin antibody. asTF- and LZ-TF-treatment of MVEC promoted adhesion of peripheral blood mononuclear cells (PBMC) under orbital shear conditions and under laminar flow; asTF-elicited adhesion was more pronounced than that elicited by LZ-TF. Expression profiling and western blotting revealed a broad activation of cell adhesion molecules (CAMs) in MVEC following asTF treatment including E-selectin, ICAM-1, and VCAM-1. In transwell assays, asTF potentiated PMBC migration through MVEC monolayers by ~3 fold under MCP-1 gradient. Conclusions TF splice variants ligate β1 integrins on MVEC, which induces the expression of CAMs in MVEC and leads to monocyte adhesion and transendothelial migration. asTF appears more potent than flTF in eliciting these effects. Our findings underscore the pathophysiologic significance of non-proteolytic, integrin-mediated signaling by the two naturally occurring TF variants in cancer and atherosclerosis.

Summary Approximately 15% of patients undergoing non‐myeloablative allogeneic haematopoietical cell transplantation (NMHCT) develop steroid‐refractory acute‐graft versus host disease (aGVHD), a usually fatal complication. We encountered 18 cases of steroid‐refractory aGVHD in 146 patients, undergoing NMHCT from a related human leucocyte antigen‐compatible donor following cyclophosphamide/fludarabine‐based conditioning. Our initial cohort of steroid‐refractory aGVHD patients treated with antithymocyte globulin (ATG) and mycophenolate mofetil (regimen‐1: n = 6) had high GVHD‐related mortality. Therefore, we investigated an alternative strategy for subsequent patients developing this complication (regimen‐2: n = 12), consisting of daclizumab (alone or combined with infliximab/ATG) and targeted broad spectrum antibacterial and aspergillus prophylaxis in conjunction with rapid tapering of steroids to minimize opportunistic infections. In a retrospective analysis, patients receiving regimen‐2 were significantly more likely to have complete resolution of GVHD compared with those receiving regimen‐1 [12/12 (100%) vs. 1/6 (17%); P < 0·001]. When compared with those receiving regimen‐1, regimen‐2 patients also had a higher probability of survival at day 100 (100% vs. 50%) and day 200 (73% vs. 17%) post‐transplant, and improved overall survival (median 453 d vs. 42 d from aGVHD onset; P < 0·0001). GVHD‐related mortality was 89% for regimen‐1 patients vs. 17% for regimen‐2 patients (P < 0·0001). These data suggest that a co‐ordinated approach using immunoregulatory monoclonal antibodies, pre‐emptive antimicrobial therapy and judicious steroid withdrawal can dramatically improve outcome in steroid‐refractory aGVHD.