Alterations in the HLA-B*57:01 Immunopeptidome by Flucloxacillin and Immunogenicity of Drug-Haptenated Peptides.

Neoantigen formation due to the interaction of drug molecules with human leukocyte antigen (HLA)-peptide complexes can lead to severe hypersensitivity reactions. Flucloxacillin (FLX), a ß-lactam antibiotic for narrow-spectrum gram-positive bacterial infections, has been associated with severe immune-mediated drug-induced liver injury caused by an influx of T-lymphocytes targeting liver cells potentially recognizing drug-haptenated peptides in the context of HLA-B*57:01. To identify immunopeptidome changes that could lead to drug-driven immunogenicity, we used mass spectrometry to characterize the proteome and immunopeptidome of B-lymphoblastoid cells solely expressing HLA-B*57:01 as MHC-I molecules. Selected drug-conjugated peptides identified in these cells were synthesized and tested for their immunogenicity in HLA-B*57:01-transgenic mice. T cell responses were evaluated in vitro by immune assays. The immunopeptidome of FLX-treated cells was more diverse than that of untreated cells, enriched with peptides containing carboxy-terminal tryptophan and FLX-haptenated lysine residues on peptides. Selected FLX-modified peptides with drug on P4 and P6 induced drug-specific CD8+ T cells in vivo. FLX was also found directly linked to the HLA K146 that could interfere with KIR-3DL or peptide interactions. These studies identify a novel effect of antibiotics to alter anchor residue frequencies in HLA-presented peptides which may impact drug-induced inflammation. Covalent FLX-modified lysines on peptides mapped drug-specific immunogenicity primarily at P4 and P6 suggesting these peptide sites as drivers of off-target adverse reactions mediated by FLX. FLX modifications on HLA-B*57:01-exposed lysines may also impact interactions with KIR or TCR and subsequent NK and T cell function.

A transgenic mouse model for HLA-B*57:01-linked abacavir drug tolerance and reactivity.

Adverse drug reactions (ADRs) are a major obstacle to drug development, and some of these, including hypersensitivity reactions to the HIV reverse transcriptase inhibitor abacavir (ABC), are associated with HLA alleles, particularly HLA-B*57:01. However, not all HLA-B*57:01+ patients develop ADRs, suggesting that in addition to the HLA genetic risk, other factors may influence the outcome of the response to the drug. To study HLA-linked ADRs in vivo, we generated HLA-B*57:01-Tg mice and show that, although ABC activated Tg mouse CD8+ T cells in vitro in a HLA-B*57:01-dependent manner, the drug was tolerated in vivo. In immunocompetent Tg animals, ABC induced CD8+ T cells with an anergy-like phenotype that did not lead to ADRs. In contrast, in vivo depletion of CD4+ T cells prior to ABC administration enhanced DC maturation to induce systemic ABC-reactive CD8+ T cells with an effector-like and skin-homing phenotype along with CD8+ infiltration and inflammation in drug-sensitized skin. B7 costimulatory molecule blockade prevented CD8+ T cell activation. These Tg mice provide a model for ABC tolerance and for the generation of HLA-B*57:01-restricted, ABC-reactive CD8+ T cells dependent on both HLA genetic risk and immunoregulatory host factors.

The ribosome destabilizes native and non-native structures in a nascent multidomain protein.

Correct folding is a prerequisite for the biological activity of most proteins. Folding has largely been studied using in vitro refolding assays with isolated small, robustly folding proteins. A substantial fraction of all cellular proteomes is composed of multidomain proteins that are often not amenable to this approach, and their folding remains poorly understood. These large proteins likely begin to fold during their synthesis by the ribosome, a large molecular machine that translates the genetic code. The ribosome affects how folding proceeds, but the underlying mechanisms remain largely obscure. We have utilized optical tweezers to study the folding of elongation factor G, a multidomain protein composed of five domains. We find that interactions among unfolded domains interfere with productive folding in the full-length protein. The N-terminal G-domain constitutes an independently folding unit that, upon in vitro refolding, adopts two similar states that correspond to the natively folded and a non-native, possibly misfolded structure. The ribosome destabilizes both of these states, suggesting a mechanism by which terminal misfolding into highly stable, non-native structures is avoided. The ribosome may thus directly contribute to efficient folding by modulating the folding of nascent multidomain proteins.

Effector functions of memory CTLs can be affected by signals received during reactivation.

Memory cytotoxic T lymphocytes (CTLs) are able to provide protections to the host against repeated insults from intracellular pathogens. However, it has not been completely understood how the effector functions of memory CTLs are induced upon antigen challenge, which is directly related to the efficacy of their protection. Third signal cytokines, such as IL-12 and type I interferon, have been suggested to be involved in the protective function of memory CTLs, but direct evidence is warranted. In this report, we found that memory CTLs need to be reactivated to exert effector functions. Infusion of a large population of quiescent memory CTLs did not lead to cancer control in tumor-bearing mice, whereas infusion of a reactivated memory CTL population did. This reactivation of memory CTLs requires cytokines such as IL-12 in addition to antigen but was less dependent upon costimulation and IL-2 compared to naive CTLs. Memory CTLs responded more quickly and with greater strength than their naive counterparts upon stimulation, which is associated with higher upregulation of important transcription factors such as T-bet and phosphorylated STAT4. In addition, memory CTLs underwent less expansion than naive CTLs upon pathogen challenge. In conclusion, effector functions of established memory CTLs may be affected by certain cytokines such as IL-12 and type I IFN. Thus, a pathogen's ability to induce cytokines could contribute to the efficacy of protection of an established memory CTL population.

Opposing impacts on healthspan and longevity by limiting dietary selenium in telomere dysfunctional mice.

Selenium (Se) is a trace metalloid essential for life, but its nutritional and physiological roles during the aging process remain elusive. While telomere attrition contributes to replicative senescence mainly through persistent DNA damage response, such an aging process is mitigated in mice with inherently long telomeres. Here, weanling third generation telomerase RNA component knockout mice carrying short telomeres were fed a Se-deficient basal diet or the diet supplemented with 0.15 ppm Se as sodium selenate to be nutritionally sufficient throughout their life. Dietary Se deprivation delayed wound healing and accelerated incidence of osteoporosis, gray hair, alopecia, and cataract, but surprisingly promoted longevity. Plasma microRNA profiling revealed a circulating signature of Se deprivation, and subsequent ontological analyses predicted dominant changes in metabolism. Consistent with this observation, dietary Se deprivation accelerated age-dependent declines in glucose tolerance, insulin sensitivity, and glucose-stimulated insulin production in the mice. Moreover, DNA damage and senescence responses were enhanced and Pdx1 and MafA mRNA expression were reduced in pancreas of the Se-deficient mice. Altogether, these results suggest a novel model of aging with conceptual advances, whereby Se at low levels may be considered a hormetic chemical and decouple healthspan and longevity.

INDUCTION OF CYTOKINE PRODUCTION IN CHEETAH (ACINONYX JUBATUS) PERIPHERAL BLOOD MONONUCLEAR CELLS AND VALIDATION OF FELINE-SPECIFIC CYTOKINE ASSAYS FOR ANALYSIS OF CHEETAH SERUM.

Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of cheetahs (Acinonyx jubatus ; n=3) and stimulated with lipopolysaccharides (LPS) to induce the production of proinflammatory cytokines TNF-α, IL-1ß, and IL-6 for establishment of cross-reactivity between these cheetah cytokines and feline-specific cytokine antibodies provided in commercially available Feline DuoSet® ELISA kits (R&D Systems, Inc., Minneapolis, Minnesota 55413, USA). This study found that feline-specific cytokine antibodies bind specifically to cheetah proinflammatory cytokines TNF-α, IL-1ß, and IL-6 from cell culture supernatants. The assays also revealed that cheetah PBMCs produce a measurable, cell concentration-dependent increase in proinflammatory cytokine production after LPS stimulation. To enable the use of these kits, which are designed for cell culture supernatants for analyzing cytokine concentrations in cheetah serum, percent recovery and parallelism of feline cytokine standards in cheetah serum were also evaluated. Cytokine concentrations in cheetah serum were approximated based on the use of domestic cat standards in the absence of cheetah standard material. In all cases (for cytokines TNF-α, IL-1ß, and IL-6), percent recovery increased as the serum sample dilution increased, though percent recovery varied between cytokines at a given dilution factor. A 1:2 dilution of serum resulted in approximately 45, 82, and 7% recovery of TNF-α, IL-1ß, and IL-6 standards, respectively. Adequate parallelism was observed across a large range of cytokine concentrations for TNF-α and IL-1ß; however, a significant departure from parallelism was observed between the IL-6 standard and the serum samples (P=0.004). Therefore, based on our results, the Feline DuoSet ELISA (R&D Systems, Inc.) kits are valid assays for the measurement of TNF-α and IL-1ß in cheetah serum but should not be used for accurate measurement of IL-6.

Transcriptome profiling of CTLs regulated by rapamycin using RNA-Seq.

Memory programming of cytotoxic T cells (CTLs) by inflammatory cytokines can be regulated by mammalian target of rapamycin (mTOR). We have shown that inhibition of mTOR during CTL activation leads to the enhancement of memory, but the molecular mechanisms remain largely unknown. Using high-throughput RNA-Seq, we identified genes and functions in mouse CTLs affected by mTOR inhibition through rapamycin. Of the 43,221 identified transcripts, 184 transcripts were differentially expressed after rapamycin treatment, corresponding to 128 annotated genes. Of these genes, 114 were downregulated and only 14 were upregulated. Most importantly, 50 of them are directly related to cell death and survival. In addition, several genes such as CD62L are related to migration. Furthermore, we predicted downregulation of transcriptional regulators based on the total differentially expressed genes, as well as the subset of apoptosis-related genes. Quantitative PCR confirmed the differential expressions detected in RNA-Seq. We conclude that the regulatory function of rapamycin may work through inhibition of multiple genes related to apoptosis and migration, which enhance CTL survival into memory.

Nicotine inhibits memory CTL programming.

Nicotine is the main tobacco component responsible for tobacco addiction and is used extensively in smoking and smoking cessation therapies. However, little is known about its effects on the immune system. We confirmed that multiple nicotinic receptors are expressed on mouse and human cytotoxic T lymphocytes (CTLs) and demonstrated that nicotinic receptors on mouse CTLs are regulated during activation. Acute nicotine presence during activation increases primary CTL expansion in vitro, but impairs in vivo expansion after transfer and subsequent memory CTL differentiation, which reduces protection against subsequent pathogen challenges. Furthermore, nicotine abolishes the regulatory effect of rapamycin on memory CTL programming, which can be attributed to the fact that rapamycin enhances expression of nicotinic receptors. Interestingly, naïve CTLs from chronic nicotine-treated mice have normal memory programming, which is impaired by nicotine during activation in vitro. In conclusion, simultaneous exposure to nicotine and antigen during CTL activation negatively affects memory development.

Amyloid peptide enhances nail rusting: novel insight into mechanisms of aging and Alzheimer's disease.

Oxidative stress is believed to play a major role in the dysfunction and degeneration of neurons that occurs in Alzheimer's disease (AD). Amyloid beta-peptide forms insoluble aggregates in the brains of AD patients and it has been shown that the neurotoxic actions of amyloid beta-peptide involve membrane lipid peroxidation. However, it is not known how amyloid beta-peptide induces oxidative stress. Here we describe a simple experiment that we performed 6 years ago that demonstrates that amyloid beta-peptide is itself a source of oxyradicals. The weights of iron nails were recorded and the nails were then incubated in one of three different solutions: water (control), 1mM amyloid beta-peptide (1-40) in water, and 1mM bovine serum albumin in water. After 1 month of incubation the nails were then removed, allowed to dry, and then their weights determined. The weights of all the nails decreased, but the amount of weight decrease in the nails that had been incubated in the presence of amyloid beta-peptide was approximately twice that of the nails incubated in the control solutions. These data provide direct evidence that amyloid beta-peptide generates, or facilitates the production of, oxyradicals thereby enhancing metal oxidation.