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Summary Herein, we demonstrate that HCMV miR-UL112-5p targets ERAP1, thereby inhibiting the processing and presentation of the HCMV pp65 495-503 peptide to specific CTLs. In addition, we show that the rs17481334 G variant, naturally occurring in the ERAP1 3′ UTR, preserves ERAP1 from miR-UL112-5p-mediated degradation. Specifically, HCMV miR-UL112-5p binds the 3′ UTR of ERAP1 A variant, but not the 3′ UTR of ERAP1 G variant, and, accordingly, ERAP1 expression is reduced both at RNA and protein levels only in human fibroblasts homozygous for the A variant. Consistently, HCMV-infected GG fibroblasts were more efficient in trimming viral antigens and being lysed by HCMV-peptide-specific CTLs. Notably, a significantly decreased HCMV seropositivity was detected among GG individuals suffering from multiple sclerosis, a disease model in which HCMV is negatively associated with adult-onset disorder. Overall, our results identify a resistance mechanism to HCMV miR-UL112-5p-based immune evasion strategy with potential implications for individual susceptibility to infection and other diseases.

Mesenchymal stromal cells (MSCs) represent a key component of bone marrow (BM) microenvironment and display immune-regulatory properties. We performed a detailed analysis of biological/functional properties of BM-MSCs derived from 33 pediatric patients affected by primary immune-deficiencies (PID-MSCs): 7 Chronic Granulomatous Disease (CGD), 15 Wiskott-Aldrich Syndrome (WAS), 11 Severe Combined Immunodeficiency (SCID). Results were compared with MSCs from 15 age-matched pediatric healthy-donors (HD-MSCs). Clonogenic and proliferative capacity, differentiation ability, immunophenotype, immunomodulatory properties were analyzed. WB and RT-qPCR for CYBB, WAS and ADA genes were performed. All PID-MSCs displayed clonogenic and proliferative capacity, morphology and immunophenotype comparable with HD-MSCs. PID-MSCs maintained the inhibitory effect on T- and B-lymphocyte proliferation, except for decreased inhibitory ability of SCID-MSCs at MSC:PBMC ratio 1:10. While HD- and CGD-MSCs were able to inhibit monocyte maturation into immature dendritic cells, in SCID- and WAS-MSCs this ability was reduced. After Toll-like Receptor priming, PID-MSCs displayed in vitro an altered gene expression profile of pro- and anti-inflammatory soluble factors. PID-MSCs displayed lower PPARγ levels and WAS- and SCID-MSCs higher levels of key osteogenic markers, as compared with HD-MSCs. Our results indicate that PID-MSCs may be defective in some functional abilities; whether these defects contribute to disease pathophysiology deserves further investigation.

Background Virus-specific CD4 and CD8 T lymphocytes from HLA-matched donors are effective for treatment and prophylaxis of viral infections in immune-compromised recipients of hematopoietic stem cell transplant recipients. Adoptive immune reconstitution is based on selection of specific T cells or on generation of specific T-cell lines from the graft donor. Unfortunately, the graft donor is not always immune to the relevant pathogen or the graft donor may not be available (registry-derived or cord blood donors). Study Design and Methods Since the possibility of using T cells from a third-party subject is now established, we screened potential donors for T-cell responses against cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus, the viruses most frequently targeted by adoptive immune reconstitution. Specific T-cell responses against viral antigens were analyzed in 111 donors using a miniaturized interferon-γ release assay. Results Responders to CMV were 64%, to EBV 40%, and to adenovirus 51%. Simultaneous responders to the three viruses were 49%. CMV-specific CD4 and CD8 T-cell lines could be generated from 11 of 12 donors defined as positive responders according to the T-cell assay. Conclusions These data demonstrate that a large fraction of volunteers can be recruited in a donor registry for selection or expansion of virus specific T cells and that our T-cell assay predicts the donors' ability to give rise to established T-cell lines endowed with proliferative potential and effector function for adoptive immune reconstitution.

Background Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation. Methods and Principal Findings Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth. Conclusions These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.

In the current study, we have investigated the effect of CB 2 and TRPV 1 receptor ligands on in vitro osteoblasts from bone marrow of human healthy donors. A pivotal role for the endocannabinoid/endovanilloid system in bone metabolism has been highlighted. We have demonstrated a functional cross-talk between CB 2 and TRPV 1 in human osteoclasts, suggesting these receptors as new pharmacological target for the treatment of bone resorption disease as osteoporosis. Moreover, we have shown the presence of these receptors on human mesenchimal stem cells, hMSCs. Osteoblasts are mononucleated cells originated from hMSCs by the essential transcription factor runt-related transcription factor 2 and involved in bone formation via the synthesis and release of macrophage colony-stimulating factor, receptor activator of nuclear factor kappa-B ligand and osteoprotegerin. For the first time, we show that CB 2 and TRPV 1 receptors are both expressed on human osteoblasts together with enzymes synthesizing and degrading endocannabinoids/endovanilloids, and oppositely modulate human osteoblast activity in culture in a way that the CB 2 receptor stimulation improves the osteogenesis whereas TRPV 1 receptor stimulation inhibits it.

Mesenchymal stromal cells (MSCs) are multipotent cells that possess broad immunomodulatory properties; the mechanisms underlying these properties have not been completely clarified. Aim of this study was to compare in vitro immunomodulatory effects of MSCs with those of microvesicles (MVs) released in supernatants from the same MSCs. MSCs were generated from bone marrow of 12 healthy donors (HDs) and MVs were isolated from their supernatant by serial ultracentrifugation according to two different procedures. Both MSCs and MVs were characterized by flow cytometry and incubated in vitro with peripheral blood mononuclear cells (PBMCs) of 12 HDs after stimulation with PHA and CpG. Growth factors and cytokines were quantified by ELISA. MVs were identified as 0.1-1μm particles positive for CMFDA, CD107, and CD13. MSCs were significantly more capable to inhibit in vitro PHA-induced T-cell proliferation as compared with the corresponding MVs (P

Alterations in hematopoietic microenvironment of acute lymphoblastic leukemia patients have been claimed to occur, but little is known about the components of marrow stroma in these patients. In this study, we characterized mesenchymal stromal cells (MSCs) isolated from bone marrow (BM) of 45 pediatric patients with acute lymphoblastic leukemia (ALL-MSCs) at diagnosis (day+0) and during chemotherapy treatment (days: +15; +33; +78), the time points being chosen according to the schedule of BM aspirates required by the AIEOP-BFM ALL 2009 treatment protocol. Morphology, proliferative capacity, immunophenotype, differentiation potential, immunomodulatory properties and ability to support long-term hematopoiesis of ALL-MSCs were analysed and compared with those from 41 healthy donors (HD-MSCs). ALL-MSCs were also genetically characterized through array-CGH, conventional karyotyping and FISH analysis. Moreover, we compared ALL-MSCs generated at day+0 with those isolated during chemotherapy. Morphology, immunophenotype, differentiation potential and in vitro life-span did not differ between ALL-MSCs and HD-MSCs. ALL-MSCs showed significantly lower proliferative capacity (p