The HLA Ligandome Comprises a Limited Repertoire of O-GlcNAcylated Antigens Preferentially Associated With HLA-B*07:02

Mass-spectrometry based immunopeptidomics has provided unprecedented insights into antigen presentation, not only charting an enormous ligandome of self-antigens, but also cancer neoantigens and peptide antigens harbouring post-translational modifications. Here we concentrate on the latter, focusing on the small subset of HLA Class I peptides (less than 1%) that has been observed to be post-translationally modified (PTM) by a O-linked N-acetylglucosamine (GlcNAc). Just like neoantigens these modified antigens may have specific immunomodulatory functions. Here we compiled from literature, and a new dataset originating from the JY B cell lymphoblastoid cell line, a concise albeit comprehensive list of O-GlcNAcylated HLA class I peptides. This cumulative list of O-GlcNAcylated HLA peptides were derived from normal and cancerous origin, as well as tissue specimen. Remarkably, the overlap in detected O-GlcNAcylated HLA peptides as well as their source proteins is strikingly high. Most of the O-GlcNAcylated HLA peptides originate from nuclear proteins, notably transcription factors. From this list, we extract that O-GlcNAcylated HLA Class I peptides are preferentially presented by the HLA-B*07:02 allele. This allele loads peptides with a Proline residue anchor at position 2, and features a binding groove that can accommodate well the recently proposed consensus sequence for O-GlcNAcylation, P(V/A/T/S)g(S/T), essentially explaining why HLA-B*07:02 is a favoured binding allele. The observations drawn from the compiled list, may assist in the prediction of novel O-GlcNAcylated HLA antigens, which will be best presented by patients harbouring HLA-B*07:02 or related alleles that use Proline as anchoring residue.

Hsa-miR-5581-3p and hsa-miR-542-3p target the F8 gene in hemophilia A without F8 mutations

Objective: This study aims at uncovering the effects of microRNAs (miRNAs) on F8 gene and FVIII protein in hemophilia A (HA). Methods: F8-targeting miRNAs were predicted by TargetScan, miRDB and starBase. MiRNAs predicted by at least two of the three databases were selected for further study, and their expressions in the blood of HA patients without F8 mutations and healthy controls were detected. Dual-luciferase reporter assay was performed to verify the binding between hsa-miR-5581-3p/hsa-miR-542-3p and F8. The regulation of F8 by hsa-miR-5581-3p/hsa-miR-542-3p was investigated in human umbilical vein endothelial cells (HUVECs) and lymphoblastoid cell line (LCL) that displayed endogenous expression of FVIII. qRT-PCR was used to detect the expressions of miRNAs and F8 gene, and Western blotting was conducted to measure the expression of FVIII protein. Results: A total of 43 F8-targeting miRNAs were predicted by at least two of the three databases. Among these miRNAs, hsa-miR-5581-3p and hsa-miR-542-3p were highly expressed in the blood of HA patients and have not been reported in previous studies of HA. Both hsa-miR-5581-3p and hsa-miR-542-3p could bind the 3’UTR of F8 mRNA. Upregulation of hsa-miR-5581-3p or hsa-miR-542-3p suppressed the expressions of F8 mRNA and FVIII protein in HUVECs and LCL cells. Conclusion: Hsa-miR-5581-3p and hsa-miR-542-3p target the F8 gene and suppress the expression of FVIII protein, which may contribute to the development of HA without F8 mutations.

Mosaic IL6ST variant inducing constitutive GP130 cytokine receptor signaling as a cause of neonatal onset immunodeficiency with autoinflammation and dysmorphy

IL6ST encodes the GP130 protein which transduces the proinflammatory signaling of the IL6 cytokine family through JAK/STAT activation. Biallelic loss-of-function IL6ST variants cause autosomal recessive hyper-IgE syndrome or a variant of Stuve-Wiedemann syndrome. Somatic gain-of-function IL6ST mutations, in particular small monoallelic in-frame deletions of which the most prevalent is IL6ST Ser187_Tyr190del, are an established cause of inflammatory hepatocellular tumors (IHCA) but so far, no disease caused by such mutations present constitutively has been described. Herein, we report a pediatric proband with a novel syndrome of neonatal onset immunodeficiency with autoinflammation and dysmorphy associated with the IL6ST Tyr186_Tyr190del variant present constitutively. Tyr186_Tyr190del was found by exome sequencing and shown to be de novo (absent in proband’s parents and siblings) and mosaic (present in approx. 15–40% of cells depending on the tissue studied—blood, urine sediment, hair bulbs, buccal swab). Functional studies were performed in EBV-immortalized patient’s B-cell lymphoblastoid cell line, which carried the variant in approximately 95% of cells. Western blot showed that the patient’s cells exhibited constitutive hyperphosphorylation of Tyr705 in STAT3 indicative of IL6 independent activation of GP130. Interestingly, the STAT3 phosphorylation could be inhibited with ruxolitinib as well as tofacitinib, which are clinically approved JAK1 and JAK3 (to lesser extent JAK2 and JAK1) inhibitors, respectively. Given our results and the recent reports of ruxolitinib and tofacitinib use for the treatment of diseases caused by direct activation of STAT3 or STAT1 we speculate that these drugs may be effective in the treatment of our patient’s condition.

In Vitro Modulation of Endogenous Antioxidant Enzyme Activities and Oxidative Stress in Autism Lymphoblastoid Cell Line (ALCL) by Stingless Bee Honey Treatment

Autism has been associated with a low antioxidant defense mechanism, while honey has been known for decades for its antioxidant and healing properties. Determination of stingless bee honey (KH) effects on antioxidant enzyme activities and oxidative damage in Autism Lymphoblastoid Cell Line (ALCL) was performed. ALCL and its normal sibling pair (NALCL) were cultured in RPMI-1640 medium at 37°C and 5% CO2. ALCL was treated with 400 μg/mL KH (24 h), and oxidative stress marker, malondialdehyde (MDA), and antioxidant enzyme activities (catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD)) were measured via enzyme-linked immunosorbent assay (ELISA), while deoxyribonucleic acid (DNA) damage was determined via comet assay. Low SOD activity ( p < 0.05 ) and high MDA level ( p < 0.05 ) were observed in ALCL compared to NALCL. Higher grade (Grades 2 and 3) of DNA damage was highly observed ( p < 0.05 ) in ALCL compared to NALCL, whereas lower grade (Grades 0 and 1) DNA damage was highly detected ( p < 0.05 ) in NALCL compared to ALCL. KH treatment caused a significant increase in SOD and GPx activities ( p < 0.05 ) in ALCL compared to untreated ALCL. Correspondingly, KH treatment reduced the Grade 2 DNA damage ( p < 0.05 ) in ALCL compared to untreated ALCL. CAT activity showed no significant difference between all three groups, while the MDA level showed no significant difference between treated and untreated ALCL. In conclusion, KH treatment significantly reduced the oxidative stress in ALCL by increasing the SOD and GPx antioxidant enzyme activities, while reducing the DNA damage.