Modulation of the Type I Interferon Response Defines the Sensitivity of Human Melanoma Cells to Oncolytic Measles Virus.

BACKGROUND: Oncolytic viruses such as live-attenuated, vaccine strains of measles virus (MV) have recently emerged as promising cancer treatments, having shown significant antitumor activity against a large variety of human tumors. OBJECTIVE: Our study aims at determining which parameters define the sensitivity of human melanoma cells to oncolytic MV infection. METHODS: We analyzed both in vitro and in vivo the oncolytic activity of MV against a panel of human melanoma cell established in our laboratory. We tested whether either type I interferons or the interferon pathway inhibitor Ruxolitinib could modulate the sensitivity of these cells to oncolytic MV infection. RESULTS: Human melanoma cells exhibit varying levels of sensitivity to MV infection in culture and as tumor xenografts. As these differences are not explained by their expression level of the CD46 receptor, we hypothesized that antiviral immune responses may be suppressed in certain cell resulting in their inability to control infection efficiently. By analyzing the type I IFN response, we found that resistant cells had a fully functional pathway that was activated upon MV infection. On the contrary, sensitive cell showed defects in this pathway. When pre-treated with IFN-α and IFN-ß, all but one of the sensitive cell became resistant to MV. Cells resistant to MV were rendered sensitive to MV with Ruxolitinib. CONCLUSION: Type I interferon response is the main determinant for the sensitivity or resistance of melanoma to oncolytic MV infection. This will have to be taken into account for future clinical trials on oncolytic MV.

Do kidney histology lesions predict long-term kidney function after liver transplantation?

Histological renal lesions observed after liver transplantation are complex, multifactorial, and interrelated. The aims of this study were to determine whether kidney lesions observed at five yr after liver transplantation can predict long-term kidney function. Ninety-nine liver transplant patients receiving calcineurin inhibitor (CNI)-based immunosuppression, who had undergone a kidney biopsy at 60±48 months post-transplant, were included in this follow-up study. Kidney biopsies were scored according to the Banff classification. Estimated glomerular filtration rate (eGFR) was assessed at last follow-up, that is, 109±48 months after liver transplantation. eGFR decreased from 92±33 mL/min at transplantation to 63±19 mL/min after six months, to 57±17 mL/min at the kidney biopsy, to 54±24 mL/min at last follow-up (p<0.0001). At last follow-up, only three patients required renal replacement therapy. After the kidney biopsy, 13 patients were converted from CNIs to mammalian target of rapamycin inhibitors, but no significant improvement in eGFR was observed after conversion. Elevated eGFR at six months post-transplant and a lower fibrous intimal thickening score (cv) observed at five yr post-transplant were the two independent predictive factors for eGFR≥60 mL/min at nine yr post-transplant. Long-term kidney function seems to be predicted by the kidney vascular lesions.

Purification of circulating plasmacytoid dendritic cells using counterflow centrifugal elutriation and immunomagnetic beads.

BACKGROUND AIMS: Plasmacytoid dendritic cells (pDC) are a dendritic cell (DC) subset specialized in the production of high amounts of interferon (IFN) type I (IFN-α, -ß) in response to viruses. They can be purified from peripheral blood mononuclear cells (PBMC), usually using magnetic bead sorting. METHODS: In this study, we set up a counterflow centrifugal elutriation (CCE) procedure to enrich pDC from PBMC. We first analyzed each CCE fraction for the presence of pDC using CD123 and BDCA-2 as markers. We then purified pDC using CCE and magnetic beads and verified that their functions were not affected by this procedure. RESULTS: pDC were sorted by CCE into intermediate fractions between those containing lymphocytes and monocytes. The pDC frequency in these intermediate fractions was 3-fold that in PBMC. Using negative-magnetic bead sorting, starting with the same number of cells and beads, we obtained more than twice as many pDC from intermediate fractions as from PBMC. The phenotypes and IFN-α production capacities of sorted pDC from PBMC and from intermediate fractions were similar, both immediately after sorting and after stimulation with CpG-A oligodeoxynucleotides. In addition, we showed that intermediate fractions could be cryopreserved and that magnetic bead sorting could be performed with the same efficiency after thawing. CONCLUSIONS: Altogether, our results show that CCE can be used to enrich lymphocytes, monocytes and pDC from the same donor, without magnetic beads on their surface. Our method should be useful for the purification of these cells for experimental research and may also be adaptable for clinical use in immunotherapy.

Downregulation of MUC1 expression and its recognition by CD8⁺ T cells on the surface of malignant pleural mesothelioma cells treated with HDACi.

Research into new treatments against malignant pleural mesothelioma (MPM) is of great interest, as this aggressive cancer is often resistant to conventional therapies. One potential strategy is the use of epigenetic drugs, such as 5-aza-2'-deoxycytidine (5-azaCdR), a DNA-hypomethylating drug, and valproate (VPA), a histone deacetylase inhibitor (HDACi). Indeed, these drugs not only trigger MPM cell death, but also induce the expression of cancer testis antigens recognized by CD8(+) T cells, such as New York-esophageal cancer-1 (NY-ESO-1). The objective of this study was to assess effects of these drugs on the expression and recognition by CD8(+) T cells of Mucin1 (MUC1), a tumor-associated antigen that is overexpressed by MPM. MPM tumor cell lines were treated with epigenetic drugs, alone or in combination. MUC1 expression by MPM cells, and its recognition by a MUC1-specific CD8(+) T-cell clone, was downregulated by HDACi when used alone or in combination with 5-azaCdR. This effect was not due to a blocking of the HLA class I presentation pathway in treated MPM cells, as NY-ESO-1 induced by 5-azaCdR alone, or with VPA, was recognized by a NY-ESO-1-specific T-cell clone. This study suggests that the choice of tumor antigens could be critical for strategies combining epigenetic drugs with immunotherapy.

New computerized color image analysis for the quantification of interstitial fibrosis in renal transplantation.

BACKGROUND: Chronic allograft injury, the primary cause of late allograft failure in renal transplantation, can be diagnosed early at a preclinical stage by histopathological changes such as interstitial fibrosis (IF). Currently, assessed by semiquantitative analysis in the Banff classification, IF quantification is limited by pathologist's subjective interpretation. METHODS: We have designed algorithms dedicated to quantify IF by computerized color image analysis. This innovative and objective software automatically extracts the green areas characteristic of IF in Masson's trichrome based on color image segmentation followed by removal of nonspecific IF staining (capsula, sclerosis glomeruli and normal glomeruli, normal basement membrane) and computes an index. It also counts automatically the number of glomeruli. Sixty-seven Masson stained renal transplant biopsies at various IF stages were imaged using a digital color camera mounted on a microscope. We tested the robustness of the method against varying acquisition parameters. RESULTS: We demonstrated that the parameters do not have an impact on this quantification and that the algorithm is able to handle biopsy color variations. The intra- and interobserver reproducibility was good (P<0.003). The kappa coefficient that was performed on another set of 90 biopsies to evaluate the concordance of our method with an expert Banff quantification was 0.68, indicating a substantial agreement. Finally, the computerized IF correlated with renal function. CONCLUSION: This study demonstrates that computerized color image analysis is a reliable and reproducible method to evaluate renal IF in routine practice and in multi-centric studies.

FOXP3-enriched infiltrates associated with better outcome in renal allografts with inflamed fibrosis.

BACKGROUND: FOXP3-expressing regulatory T cells (Tregs) play a crucial role in maintaining allogeneic transplant tolerance in experimental models. In clinical transplantation, there are few data about their role in chronic inflammation. We hypothesized that Tregs might accumulate within the graft since enrichment of Tregs has been frequently described in chronically inflamed tissues. METHODS: Sixty-seven biopsies, indicated by a rise in creatinine level, were studied. Thirty-four biopsies showing acute T-cell-mediated rejection and 33 displaying inflamed fibrosis were selected. Tregs frequency was calculated for each infiltrate by counting FOXP3+ and CD3+ cells on two contiguous serial sections. RESULTS: A total of 121 infiltrates were scored with a mean of 309 CD3+ cells per infiltrate (range: 50-700). Tregs were enriched within allografts exhibiting inflamed fibrosis versus acute cellular rejection (10.6 +/- 6.8% versus 5.5 +/- 2.6%, respectively, P = 0.005). In those with inflammation within scarred areas, the subset of patients with a low FOXP3/CD3 ratio (below the median value) displayed a lower frequency of B-cell-enriched nodular cell clusters (20% versus 86%, P = 0.001) and had a significantly lower graft survival (log-rank, P = 0.02). In multivariate analysis, the low FOXP3/CD3 ratio remained an independent indicator of outcome (P = 0.03). Consistently, the FOXP3+/IL-17+ cell ratio was higher in nodular than in diffuse infiltrates. CONCLUSIONS: Our results suggest that Tregs may dampen the graft injury in chronic (versus acute) inflammation and stress the importance of devising strategies to enhance Tregs efficiency.