Physical detection of influenza A epitopes identifies a stealth subset on human lung epithelium evading natural CD8 immunity

Vaccines eliciting immunity against influenza A viruses (IAVs) are currently antibody-based with hemagglutinin-directed antibody titer the only universally accepted immune correlate of protection. To investigate the disconnection between observed CD8 T-cell responses and immunity to IAV, we used a Poisson liquid chromatography data-independent acquisition MS method to physically detect PR8/34 (H1N1), X31 (H3N2), and Victoria/75 (H3N2) epitopes bound to HLA-A*02:01 on human epithelial cells following in vitro infection. Among 32 PR8 peptides (8–10mers) with predicted IC₅₀ < 60 nM, 9 were present, whereas 23 were absent. At 18 h postinfection, epitope copies per cell varied from a low of 0.5 for M1_3–11 to a high of >500 for M1_58–66 with PA, HA, PB1, PB2, and NA epitopes also detected. However, aside from M1_58–66, natural CD8 memory responses against conserved presented epitopes were either absent or only weakly observed by blood Elispot. Moreover, the functional avidities of the immunodominant M1_58–66/HLA-A*02:01-specific T cells were so poor as to be unable to effectively recognize infected human epithelium. Analysis of T-cell responses to primary PR8 infection in HLA-A*02:01 transgenic B6 mice underscores the poor avidity of T cells recognizing M1_58–66. By maintaining high levels of surface expression of this epitope on epithelial and dendritic cells, the virus exploits the combination of immunodominance and functional inadequacy to evade HLA-A*02:01-restricted T-cell immunity. A rational approach to CD8 vaccines must characterize processing and presentation of pathogen-derived epitopes as well as resultant immune responses. Correspondingly, vaccines may be directed against “stealth” epitopes, overriding viral chicanery.Human influenza is an acute respiratory infection caused by Orthomyxoviridae, a family of single-stranded, negative sense RNA viruses containing a segmented genome. The influenza virion is enveloped by a lipid bilayer derived from the host cell with hemagglutinin (HA), neuraminidase (NA), and matrix 2 (M2) transmembrane proteins of the virus exposed and antibody (Ab) accessible. HA is the most abundant protein on the surface and is the principle antigen recognized by humoral immunity (1). RNA replication is error prone, with influenza A virus (IAV) averaging roughly one error for each replicated genome (2). Notably, the HA protein manifests a high functional tolerance to sequence variation not evident in some of the internal proteins (3). Current vaccines protect largely by inducing Abs against the HA and NA proteins but require continual reformulation based on inferring the dominant strains that will circulate in the upcoming flu season, an imperfect science at best. Recognizing the challenges for universal protection against influenza via antibodies, there has been extensive discussion about vaccines harnessing cellular immunity (4–6). Cytotoxic T lymphocytes (CTLs), primarily CD8⁺ T cells, can recognize and kill infected cells when their T-cell receptors (TCRs) recognize fragments of viral proteins that are in turn bound to major histocompatibility complex (MHC in general; for humans, HLA) proteins on the surface of infected cells. These peptides can derive from segments of internal proteins conserved among IAV strains so that CTLs targeting these sequence-constrained peptides would remain effective as surface HA and NA antigens, in contrast, change by genetic drift or shift. There is substantial evidence for T-cell–mediated protection in mice (7, 8), but qualified evidence in humans (9, 10). The limited CTL protection has been ascribed to slow cellular responses against IAV, a latency reflecting the expansion and activation of central memory T cells in lymph nodes and the subsequent recruitment of CTLs to infected lung (11). The recent identification of CD8⁺ effector resident memory T cells (T_RM) within barrier tissues postinfection (12, 13) could challenge this picture. However, vaccines for cellular immunity are at an early stage of development and what formulations and delivery methods are needed to harness T_RM immunity is not well understood. In particular, the pathogen-derived peptides bound to HLA molecules on the surface of IAV-infected lung epithelial cells are unknown. As a T cell monitors a single MHC-restricted antigen, surveillance is constrained by the numbers of antigen-specific cells and their motility in lung parenchyma under homeostatic conditions. Sentinel T_RM cell populations lodged in barrier tissues are limited, so that inducing irrelevant specificities will necessarily displace those with useful specificities. In contrast, the fluid volume surrounding a cell at the site of infection can contain a large set of antibodies recognizing diverse antigens. Rational T-cell vaccine development must focus on the peptides directly presented by infected lung epithelium and identify those peptides that can be recognized by high avidity T_RM cells.IAV peptides displayed by infected cells are conventionally identified by T-cell functional assays. In principle this “reverse immunology” only identifies what epitopes the antigen-experienced host has recognized, not what can or should be recognized. As such, reverse immunology is a poor guide for vaccine development. Here new acquisition and analysis methods in mass spectrometry (MS) are applied to directly identify these peptides. New methodology is necessary as conventional data-dependent acquisition (DDA) MS, identifying ions largely in order of signal intensity, cannot plow deep enough into the sample to uncover the IAV peptides underneath an ocean of “self” peptides. Reflecting the prevalence of the HLA-A2 allele in the population and the large number of studies of IAV infection and CD8⁺ T-cell responses restricted by this allele, this study focuses on antigen presentation by HLA-A*02:01 (A2). Our results identify previously unrecognized IAV epitopes that may be useful in vaccine formulations, question the veracity of functionally ascribed epitope display, and show via stable isotope labeling by amino acids in culture (SILAC) that positive strand RNA-derived translation and not protein cross-presentation is the basis of A2 IAV peptidome array on dendritic cells (DCs) that phagocytose non-A2 IAV-infected UV-irradiated cells. In addition, our data rationalize why an immunodominant CD8 T-cell response to the highly conserved M1_58–66 peptide GILGFVFTL does not provide sterilizing immunity to IAV.     

Peritoneal macrophages function modulation by L-carnitine in aging rats

BACKGROUND AND AIMS: The aging process is associated with a progressive decline in physiological functions involving immune response in most species. The aim of the present study was to determine the effect of L-carnitine on impaired macrophages function in aged rats. METHODS: Superoxide anion production, chemotaxis and phagocytic activity were studied in peritoneal macrophages obtained from young (2 months old) and aged (24 months old) rats. L-carnitine (50 mg/kg bw) or control vehicle was orally gavaged into young and aged rats for 30 consecutive days. RESULTS: The peritoneal macrophages of the aged rats exhibited an increase in superoxide anion generation and a decline in chemotaxis and phagocytic index by comparison with the young rats. Superoxide anion production in aged rats was significantly reduced by L-carnitine treatment, as accompanied by a significant enhancement of chemotactic activity, which was restored to control levels observed in young rats. The age-related reduction in phagocytic index was only slightly, but not significantly, restored by L-carnitine administration, however. CONCLUSION: The findings suggest that L-carnitine administration may be useful in reversing some age-related changes.     

Effect of L-carnitine on carrageenan-induced inflammation in aged rats

BACKGROUND: It is known that L-carnitine is a cofactor in the transport of fatty acids across the inner mitochondrial membrane for beta-oxidation. However, L-carnitine is an antioxidant compound widely used for the treatment of deficits in functions due to the aging process. OBJECTIVE: The purpose of the study was to investigate the effect of L-carnitine on carrageenan-induced inflammation in aged rats. METHODS: L-carnitine (50 mg/kg/day) or control vehicle was given by gavage for 30 consecutive days to young (2-month-old) and aged (24-month old) rats. 6 ml of air was injected subcutaneously into the dorsum of each rat, followed 2 days later by 4 ml of 2% carrageenan. After 2 days, the exudate was collected from the inflamed site of each rat. The quantity of collected exudate and the number of cells which have migrated to the inflamed site were determined. RESULTS: No differences were observed in quantity of exudate in all groups; a decrease in the number of exudate cells was established in aged rats. However, L-carnitine treatment significantly increased the number of exudate cells in both young and aged rats. The exudate cells from the aged rats exhibited a decline of both phagocytic and chemotactic activities as compared with those from the young rats, and the decreased functions were significantly enhanced by L-carnitine treatment. However, superoxide anion release was seen to be unchanged in exudate cells due to aging, and L-carnitine intake decreased the production of superoxide anion by these cells in young and aged rats. CONCLUSIONS: These findings demonstrate that L-carnitine is capable of restoring the age-related changes in the functions of inflammatory cells. Moreover, L-carnitine may play a protective role in the tissue destruction in inflammation by decreasing the superoxide anion production.     

Effect of alpha-lipoic acid on visual evoked potentials in rats exposed to sulfite

This study aimed to investigate the effect of alpha-lipoic acid (LA) administration on sulfite-induced alterations in visual evoked potentials (VEPs). Fifty two male albino Wistar rats were randomized into four experimental groups as follows; control (C), LA treated (L), sodium metabisulfite (Na(2)S(2)O(5)) treated (S), Na(2)S(2)O(5)+LA treated (SL). Na(2)S(2)O(5) (260 mg/kg/day) and LA (100 mg/kg/day) were given by intragastric intubation for 5 weeks. The latencies of VEP components were significantly prolonged in the S group and returned to control levels following LA administration. Thiobarbituric acid reactive substances (TBARS) levels in the S group were significantly higher than those detected in controls. LA significantly decreased brain and retina TBARS levels in the SL group compared with the S group. Sulfite caused a significant decrease in retina and brain glutathione peroxidase (GPx) activities which was restored to control levels via LA administration. Brain glutathione (GSH):glutathione disulfide (GSSG) ratio was significantly increased in rats jointly treated with sulfite and LA compared to rats treated with sulfite alone. Though not significant, a similar increase in GSH:GSSG ratio was also observed in the retina of SL group. This study showed that LA is protective against sulfite-induced VEP alterations and oxidative stress in the brain and retina.     

Promoting tolerance to proteolipid protein-induced experimental autoimmune encephalomyelitis through targeting dendritic cells

In T cell-mediated autoimmune diseases, self-reactive T cells with known antigen specificity appear to be particularly promising targets for antigen-specific induction of tolerance without compromising desired protective host immune responses. Several lines of evidence suggest that delivery of antigens to antigen-presenting dendritic cells (DCs) in the steady state (i.e., to immature DCs) may represent a suitable approach to induce antigen-specific T-cell tolerance peripherally. Here, we report that anti-DEC205–mediated delivery of the self-peptide proteolipid protein (PLP)139–151 to DCs ameliorated clinical symptoms in the PLP-induced SJL model of experimental autoimmune encephalomyelitis. Splenocytes from treated mice were anergized to PLP139–151, and IL-17 secretion was markedly reduced. Moreover, we show directly, using transgenic CD4⁺ Vβ6⁺ TCR T cells specific for PLP139–151, that, under the conditions of the present experiments, these cells also became anergic. In addition, evidence for a CD4⁺ T cell-mediated suppressor mechanism was obtained.     

The early kinetics of gentamicin uptake into the inner ear

Transtympanic gentamicin administration has become a popular modality in the treatment of Ménière's disease. This modality and other inner-ear medical therapy are gaining increased clinical and scientific attention. We previously described the kinetics and effects of gentamicin uptake into the inner ear after delivery of the medicine into the middle ear using a variety of different techniques and sustained-release modalities [1]. In our previous work, we reported an early peak perilymph concentration and the presence of intracellular gentamicin at the 4-hour time point. We also demonstrated the activation of inner-ear damage pathways at this early time point. In this report, we examine the kinetics of gentamicin at very early time points, 1 and 2 hours after administration. Healthy adult chinchillas underwent implantation of middle-ear sustained-release devices (one to each ear) containing gentamicin. The animals then were maintained in a neutral position and underwent perilymph gentamicin sampling at the two predetermined time points. This technique allowed us to assess accurately very early time point inner-ear gentamicin kinetics and to compare the activity. The samples then were run for concentration using mass spectrometry. The information gained from this study may increase our scientific understanding about the effects of gentamicin on the inner ear and may allow clinicians to treat patients more effectively for inner-ear disorders.     

The Effects of Macrophages on the Survival of Random Skin Flaps in Swine

Tissue macrophages play a critical role in neovascularization by releasing angiogenic cytokines. Macrophages normally arrive into a wound bed 48 to 96 hours following an injury. ^1. Introducing macrophages into a wound bed at the time of closure would theoretically stimulate neovascularization in the traumatized tissue prior to what is normally observed. The ability to promote early angiogenesis could be an important factor in the survival of an extended skin flap. By taking advantage of advanced cell-sorting techniques, the authors developed the first study to evaluate the effect of placing a purified autologous macrophage population into an extended skin flap. We created 72 dorsally based random skin flaps in Yorkshire pigs; 48 of these wounds received autologous macrophages while 24 flaps served as controls. The macrophages were obtained by utilizing monoclonal antibodies in conjunction with flow cytometry. The skin flaps were evaluated on postoperative day 6 for their viability. Analysis of the data showed no statistically significant difference between the control and treatment flaps. There was, however, a trend of increased survival for flaps treated with macrophages. This is the first study to investigate using a purified population of cells to improve the survival of random skin flaps.     

Chylothorax

During a high-speed road traffic accident, a 26-year-old man suffered multiple fractures of his thoracic vertebrae and bilateral pneumothoraces. The day after admission and commencement of nasogastric feeding, milky fluid was noted in his right chest drain. Feeding was stopped and a contrast oesophogram and oesophagoscopy were performed, which were normal. The chylothorax quickly resolved and both drains were removed on day 6. Initial treatment of chylothorax aims to decompress the pleural space and minimise chyle production by not feeding the patient via the enteral route. Most authors recommend conservative management for 2 weeks or more unless certain parameters are met: average daily chyle loss of > 1.5 l for a 5-day period, or imminent nutritional complications. In this case, surgical management of the chylothorax and spinal fractures was planned. However, conservative management was successful, highlighting the fact that early aggressive surgical intervention for chyle leaks in blunt trauma is not necessary.