Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia

Abstract Leukemias are highly immunogenic, but they have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I–associated peptide antigens with O-linked β-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these posttranslationally modified neoantigens provide logical targets for cancer immunotherapy. Cancer Immunol Res; 5(5); 376–84. ©2017 AACR.</jats:p

Anaesthetic Impairment of Immune Function Is Mediated via GABAA Receptors

GABA(A) receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A) receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients.We demonstrate, using RT-PCR, that monocytes express GABA(A) receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin.Our results show that functional GABA(A) receptors are present on monocytes with properties similar to CNS GABA(A) receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A) receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A) receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Inhibition of monocyte phagocytosis by anaesthetics is reversed by GABA_A antagonists.

<p><b>a</b>) Concentration dependent inhibition of phagocytosis by thiopental in primary human monocytes; <b>b</b>) In the presence of either propofol (PPF) or sodium thiopental (STP), phagocytosis was significantly restored by the addition of the GABA_A receptor antagonists picrotoxin (PTX) and bicuculline (BIC) (* sig diff. Mann-Whitney U test: p<0.05).</p

Inhibition of monocyte migration by anaesthetics is reversed by GABA_A antagonists.

<p><b>a</b>) Schematic representation of the <i>in vitro</i> transwell chemotaxis apparatus used to assay human primary monocyte migration; <b>b and c</b>) Concentration-dependent inhibition of chemotaxis was observed in the presence of propofol (PPF) and sodium thiopental (STP) (IC₅₀s  = 119 µM and 274 µM, respectively, n = 6); <b>d and e</b>) In the presence of either propofol (PPF) or sodium thiopental (STP), chemotaxis was significantly restored by the addition of the GABA_A antagonists picrotoxin (PTX) and bicuculline (BIC); (** sig diff. Mann-Whitney U test: p<0.01). Experiments were also performed with THP-1 cells and no significant differences between the behaviour of freshly-prepared human monocytes and THP-1 cells were seen (data not shown).</p

Functional responses of GABA_A receptors in THP-1 cells.

<p><b>a</b>) Typical whole-cell patch-clamp traces of THP-1 cells clamped at +60 mV in the presence of GABA and muscimol; currents were blocked by the GABA_A receptor antagonists bicuculline and picrotoxin. <b>b</b>) Application of muscimol (300 µM) to THP-1 cells revealed a reversal potential (E_rev) of 15.1 mV, and <b>c</b>) chloride-selectivity (P_Na/P_Cl) of 0.23 (n = 5). <b>d</b>) Typical Flexstation responses of THP-1 cells to different concentrations of muscimol. Buffer (continuous line) or muscimol (3, 10 and 100 µM, dashed lines) were added at 20 s. <b>e</b>) Dose curve response of THP-1 cells; EC₅₀  = 2.5 µM, n = 6.</p

GABA_A receptor subunits detected using RT-PCR in human monocytes, THP-1 cells and human cerebral cortex (positive control); n = 4–12.

<p>Typical immunoblots are show in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017152#pone-0017152-g001" target="_blank">figure 1</a>.</p

mRNA and protein expression of GABA_A receptor subunits in monocytic cells.

<p><b>a</b>) Typical RT-PCR of total RNA isolated from monocyte (M) and THP-1 cell (T) lysates detecting GABA_A receptor subunits. Amplimers corresponding to the expected sizes were detected for α4, γ1 and δ subunits of the GABA_A receptor in THP-1 cells, and β2 subunits in monocytes. RNA isolated from whole human brain (B) was used as a positive control. <b>b</b>) GABA_A receptor β2 subunit expression in non–permeabilised human monocytes. Image of a human monocyte stained with Hoescht 33342 (left hand panel) to show the nucleus, and with a GABA_A receptor β2-specific polyclonal antiserum (centre) revealing cell surface β2 subunits. The right panel shows the merged image. Positive controls were human cerebral cortex and negative controls were neutrophils (data not shown). Scale bar  = 5 µm. Data are typical of at least 6 independent experiments. Inset  =  typical immunoblot of a monocyte sample (left hand side) and control (neutrophil, right hand side) probed with the β2-specific antiserum and showing expected MWt for a β2 subunit.</p