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Mucopolysaccharide diseases (MPS) are caused by deficiency of glycosaminoglycan (GAG) degrading enzymes, leading to GAG accumulation. Neurodegenerative MPS diseases exhibit cognitive decline, behavioural problems and shortened lifespan. We have characterised neuropathological changes in mouse models of MPSI, IIIA and IIIB to provide a better understanding of these events.Wild-type (WT), MPSI, IIIA and IIIB mouse brains were analysed at 4 and 9 months of age. Quantitative immunohistochemistry showed significantly increased lysosomal compartment, GM2 ganglioside storage, neuroinflammation, decreased and mislocalised synaptic vesicle associated membrane protein, (VAMP2), and decreased post-synaptic protein, Homer-1, in layers II/III-VI of the primary motor, somatosensory and parietal cortex. Total heparan sulphate (HS), was significantly elevated, and abnormally N-, 6-O and 2-O sulphated compared to WT, potentially altering HS-dependent cellular functions. Neuroinflammation was confirmed by significantly increased MCP-1, MIP-1α, IL-1α, using cytometric bead arrays. An overall genotype effect was seen in all parameters tested except for synaptophysin staining, neuronal cell number and cortical thickness which were not significantly different from WT. MPSIIIA and IIIB showed significantly more pronounced pathology than MPSI in lysosomal storage, astrocytosis, microgliosis and the percentage of 2-O sulphation of HS. We also observed significant time progression of all genotypes from 4-9 months in lysosomal storage, astrocytosis, microgliosis and synaptic disorganisation but not GM2 gangliosidosis. Individual genotype*time differences were disparate, with significant progression from 4 to 9 months only seen for MPSIIIB with lysosomal storage, MPSI with astrocytocis and MPSIIIA with microgliosis as well as neuronal loss. Transmission electron microscopy of MPS brains revealed dystrophic axons, axonal storage, and extensive lipid and lysosomal storage. These data lend novel insight to MPS neuropathology, suggesting that MPSIIIA and IIIB have more pronounced neuropathology than MPSI, yet all are still progressive, at least in some aspects of neuropathology, from 4-9 months.

Aim: Assembly of HLA class I complex is assisted by the peptide loading complex and its determinant Tapasin (TPN). The crucial role of TPN in selecting peptides makes it an ideal target for viral immune escape mechanisms. For instance the immune evasion strategy of HCMV prevents the presentation of viral antigens by targeting TPN. Our recent investigations highlighted the importance of residue 156 within the PBR in determining TPN dependence of assembly and peptide binding specificities for HLA-B∗44 allotypes. We extended this work further to HLA-A∗24 allelic variants which are known to process and present the IE-1 peptides following HCMV infection. Methods: We compared the TPN dependency and peptide repertoire of A∗24:02Gln156, A∗24:06Trp156 and A∗24:13Leu156. Class I negative and class I/TPN negative cells were lentivirally transduced with HLA-A∗24 full length constructs, mRNA transcripts verified by real time PCR and surface expression of complexes assessed by flow cytometry. Peptides eluted from soluble HLA-A∗24 molecules loaded in the presence or absence of TPN were sequenced by LC-ESI-MSMS. Results: Both A∗24:06 and A∗24:13 showed high levels of surface expression (∼75%) in the absence of TPN, while A∗24:02 exhibited partial independence. Their peptides clearly showed different patterns and features. Structurally, HLA-A∗24:02 contains the residue triad Met97/His114/Gln156, while A∗24:06 and A∗24:13 contain a Trp and Leu polymorphism respectively at 156 that provides TPN independence by stabilizing the triad residues, thus generating an energetically stable and a more peptide receptive environment. Conclusions: TPN independent alleles might help to combat infections. However, presentation of unusual ligands by these alleles could be a risk factor during stem cell transplantation and needs to be considered during donor selection. The future of HSCT relies on our understanding of how successful clinical outcomes can be achieved despite patient-donor allelic mismatches. [Copyright &y& Elsevier]

Tumor-infiltrating myeloid-derived suppressor cells (MDSC) are associated with poor survival outcomes in many human cancers. MDSCs inhibit T cell-mediated tumor immunity in part because they strongly inhibit T-cell function. However, whether MDSCs inhibit early or later steps of T-cell activation is not well established. Here we show that MDSCs inhibited proliferation and induced apoptosis of CD8 + T cells even in the presence of dendritic cells (DC) presenting a high-affinity cognate peptide. This inhibitory effect was also observed with delayed addition of MDSCs to cocultures, consistent with functional data showing that T cells expressed multiple early activation markers even in the presence of MDSCs. Single-cell RNA-sequencing analysis of CD8 + T cells demonstrated a p53 transcriptional signature in CD8 + T cells cocultured with MDSCs and DCs. Confocal microscopy showed induction of DNA damage and nuclear accumulation of activated p53 protein in a substantial fraction of these T cells. DNA damage in T cells was dependent on the iNOS enzyme and subsequent nitric oxide release by MDSCs. Small molecule-mediated inhibition of iNOS or inactivation of the Nos2 gene in MDSCs markedly diminished DNA damage in CD8 + T cells. DNA damage in CD8 + T cells was also observed in KPC pancreatic tumors but was reduced in tumors implanted into Nos2 -deficient mice compared with wild-type mice. These data demonstrate that MDSCs do not block early steps of T-cell activation but rather induce DNA damage and p53 pathway activation in CD8 + T cells through an iNOS-dependent pathway., (©2021 American Association for Cancer Research.)

Background: Polymorphic differences between donor and recipient human leukocyte antigen class I molecules can result in graft-versus-host disease due to distinct peptide presentation. As part of the peptide-loading complex, tapasin plays an important role in selecting peptides from the pool of potential ligands. Class I polymorphisms can significantly alter the tapasin-mediated interaction with the peptide-loading complex and although most class I allotypes are highly dependent upon tapasin, some are able to load peptides independently of tapasin. Several human leukocyte antigen B*44 allotypes differ exclusively at position 156 (B*44:02(156Asp), 44:03(156Leu), 44:28(156Arg), 44:35(156Glu)). From these alleles, only the high tapasin-dependency of human leukocyte antigen B*44:02 has been reported., Design and Methods: We investigated the influence of position 156 polymorphisms on both the requirement of tapasin for efficient surface expression of each allotype and their peptide features. Genes encoding human leukocyte antigen B*44 variants bearing all possible substitutions at position 156 were lentivirally transduced into human leukocyte antigen class I-negative LCL 721.221 cells and the tapasin-deficient cell line LCL 721.220., Results: Exclusively human leukocyte antigen B*44:28(156Arg) was expressed on the surface of tapasin-deficient cells, suggesting that the remaining B*44/156 variants are highly tapasin-dependent. Our computational analysis suggests that the tapasin-independence of human leukocyte antigen B*44:28(156Arg) is a result of stabilization of the peptide binding region and generation of a more peptide receptive state. Sequencing of peptides eluted from human leukocyte antigen B*44 molecules by liquid chromatography-electrospray ionization-mass spectrometry (LTQ-Orbitrap) demonstrated that both B*44:02 and B*44:28 share the same overall peptide motif and a certain percentage of their individual peptide repertoires in the presence and/or absence of tapasin., Conclusions: Here we report for the first time the influence of position 156 on the human leukocyte antigen/tapasin association. Additionally, the results of peptide sequencing suggest that tapasin chaperoning is needed to acquire peptides of unusual length.