No abstract

Induction of type I IFNs is a fundamental cellular response to both viral and bacterial infection. The role of the transcription factor IRF3 is well established in driving this process. However, equally as important are cellular mechanisms for turning off type I IFN production to limit this response. In this respect, IRF3 has previously been shown to be targeted for ubiquitin-mediated degradation postviral detection to turn off the IFN-β response. In this study, we provide evidence that the E3 ligase Ro52 (TRIM21) targets IRF3 for degradation post-pathogen recognition receptor activation. We demonstrate that Ro52 interacts with IRF3 via its C-terminal SPRY domain, resulting in the polyubiquitination and proteasomal degradation of the transcription factor. Ro52-mediated IRF3 degradation significantly inhibits IFN-β promoter activity, an effect that is reversed in the presence of the proteasomal inhibitor MG132. Specific targeting of Ro52 using short hairpin RNA rescues IRF3 degradation following polyI:C-stimulation of HEK293T cells, with a subsequent increase in IFN-β production. Additionally, shRNA targeting of murine Ro52 enhances the production of the IRF3-dependent chemokine RANTES following Sendai virus infection of murine fibroblasts. Collectively, this demonstrates a novel role for Ro52 in turning off and thus limiting IRF3-dependent type I IFN production by targeting the transcription factor for polyubiquitination and subsequent proteasomal degradation.

Purpose: Combination PD-1 and CTLA-4 inhibitor therapy has dramatically improved the survival of patients with advanced melanoma but is also associated with significant immune-related toxicities. This study sought to identify circulating cytokine biomarkers of treatment response and immune-related toxicity. Experimental Design: The expression of 65 cytokines was profiled longitudinally in 98 patients with melanoma treated with PD-1 inhibitors, alone or in combination with anti-CTLA-4, and in an independent validation cohort of 49 patients treated with combination anti-PD-1 and anti-CTLA-4. Cytokine expression was correlated with RECIST response and immune-related toxicity, defined as toxicity that warranted permanent discontinuation of treatment and administration of high-dose steroids. Results: Eleven cytokines were significantly upregulated in patients with severe immune-related toxicities at base-line (PRE) and early during treatment (EDT). The expression of these 11 cytokines was integrated into a single toxicity score, the CYTOX (cytokine toxicity) score, and the predictive utility of this score was confirmed in the discovery and validation cohorts. The AUC for the CYTOX score in the validation cohort was 0.68 at PRE [95% confidence interval (CI), 0.51-0.84; P ¼ 0.037] and 0.70 at EDT (95% CI, 0.55-0.85; P ¼ 0.017) using ROC analysis. Conclusions: The CYTOX score is predictive of severe immune-related toxicity in patients with melanoma treated with combination anti-CTLA-4 and anti-PD-1 immunotherapy. This score, which includes proinflammatory cytokines such as IL1a, IL2, and IFNa2, may help in the early management of severe, potentially lifethreatening immune-related toxicity. See related commentary by Johnson and Balko, p. 1452

Although most self-reactive T cells are eliminated in the thymus, mechanisms to inactivate or control T cells specific for extrathymic antigens are required and exist in the periphery. By investigating the site in which autoreactive T cells are tolerized, we identify a unique mechanism of peripheral deletion in which naïve autoreactive CD8 T cells are rapidly eliminated in the liver after intrahepatic activation. T cells actively invade hepatocytes, enter endosomal/lysosomal compartments, and are degraded. Blockade of this process leads to accumulation of autoreactive CD8 T cells in the liver and breach of tolerance, with the development of autoimmune hepatitis. Cell into cell invasion, or emperipolesis, is a long-observed phenomenon for which a physiological role has not been previously demonstrated. We propose that this "suicidal emperipolesis" is a unique mechanism of autoreactive T-cell deletion, a process critical for the maintenance of tolerance.

No abstract

BackgroundAdipose tissue is an attractive source of cells for therapeutic purposes because of the ease of harvest and the high frequency of mesenchymal stem cells (MSCs). Whilst it is clear that MSCs have significant therapeutic potential via their ability to secrete immuno-modulatory and trophic cytokines, the therapeutic use of mixed cell populations from the adipose stromal vascular fraction (SVF) is becoming increasingly common.MethodsIn this study we have measured a panel of 27 cytokines and growth factors secreted by various combinations of human adipose-derived cell populations. These were 1. co-culture of freshly isolated SVF with adipocytes, 2. freshly isolated SVF cultured alone, 3. freshly isolated adipocytes alone and 4. adherent adipose-derived mesenchymal stem cells (ADSCs) at passage 2. In addition, we produced an ‘in silico’ dataset by combining the individual secretion profiles obtained from culturing the SVF with that of the adipocytes. This was compared to the secretion profile of co-cultured SVF and adipocytes. Two-tailed t-tests were performed on the secretion profiles obtained from the SVF, adipocytes, ADSCs and the ‘in silico’ dataset and compared to the secretion profiles obtained from the co-culture of the SVF with adipocytes. A p-value of < 0.05 was considered statistically different. To assess the overall changes that may occur as a result of co-culture we compared the proteomes of SVF and SVF co-cultured with adipocytes using iTRAQ quantitative mass spectrometry.ResultsA co-culture of SVF and adipocytes results in a distinct secretion profile when compared to all other adipose-derived cell populations studied. This illustrates that cellular crosstalk during co-culture of the SVF with adipocytes modulates the production of cytokines by one or more cell types. No biologically relevant differences were detected in the proteomes of SVF cultured alone or co-cultured with adipocytes.ConclusionsThe use of mixed adipose cell populations does not appear to induce cellular stress and results in enhanced secretion profiles. Given the importance of secreted cytokines in cell therapy, the use of a mixed cell population such as the SVF with adipocytes may be considered as an alternative to MSCs or fresh SVF alone.

High throughput identification of proteins by peptide mass fingerprinting requires an efficient means of picking peaks from mass spectra. Here, we report the development of a peak harvester to automatically pick monoisotopic peaks from spectra generated on matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometers. The peak harvester uses advanced mathematical morphology and watershed algorithms to first process spectra to stick representations. Subsequently, Poisson modelling is applied to determine which peak in an isotopically resolved group represents the monoisotopic mass of a peptide. We illustrate the features of the peak harvester with mass spectra of standard peptides, digests of gel-separated bovine serum albumin, and with Escherictia coli proteins prepared by two-dimensional polyacrylamide gel electrophoresis. In all cases, the peak harvester proved effective in its ability to pick similar monoisotopic peaks as an experienced human operator, and also proved effective in the identification of monoisotopic masses in cases where isotopic distributions of peptides were overlapping. The peak harvester can be operated in an interactive mode, or can be completely automated and linked through to peptide mass fingerprinting protein identification tools to achieve high throughput automated protein identification.

Tissue samples (plasma, saliva, serum or urine) from 169 patients classified as either normal or having one of seven possible diseases are analysed across three 96-well plates for the presences of 37 analytes using cytokine inflammation multiplexed immunoassay panels. Censoring for concentration data caused problems for analysis of the low abundant analytes. Using fluorescence analysis over concentration based analysis allowed analysis of these low abundant analytes. Mixed-effects analysis on the resulting fluorescence and concentration responses reveals a combination of censoring and mapping the fluorescence responses to concentration values, through a 5PL curve, changed observed analyte concentrations. Simulation verifies this, by showing a dependence on the mean florescence response and its distribution on the observed analyte concentration levels. Differences from normality, in the fluorescence responses, can lead to differences in concentration estimates and unreliable probabilities for treatment effects. It is seen that when fluorescence responses are normally distributed, probabilities of treatment effects for fluorescence based t-tests has greater statistical power than the same probabilities from concentration based t-tests. We add evidence that the fluorescence response, unlike concentration values, doesn’t require censoring and we show with respect to differential analysis on the fluorescence responses that background correction is not required.