A vaccine targeting resistant tumours by dual T cell plus NK cell attack.

Most cancer vaccines target peptide antigens, necessitating personalization owing to the vast inter-individual diversity in major histocompatibility complex (MHC) molecules that present peptides to T cells. Furthermore, tumours frequently escape T cell-mediated immunity through mechanisms that interfere with peptide presentation1. Here we report a cancer vaccine that induces a coordinated attack by diverse T cell and natural killer (NK) cell populations. The vaccine targets the MICA and MICB (MICA/B) stress proteins expressed by many human cancers as a result of DNA damage2. MICA/B serve as ligands for the activating NKG2D receptor on T cells and NK cells, but tumours evade immune recognition by proteolytic MICA/B cleavage3,4. Vaccine-induced antibodies increase the density of MICA/B proteins on the surface of tumour cells by inhibiting proteolytic shedding, enhance presentation of tumour antigens by dendritic cells to T cells and augment the cytotoxic function of NK cells. Notably, this vaccine maintains efficacy against MHC class I-deficient tumours resistant to cytotoxic T cells through the coordinated action of NK cells and CD4+ T cells. The vaccine is also efficacious in a clinically important setting: immunization following surgical removal of primary, highly metastatic tumours inhibits the later outgrowth of metastases. This vaccine design enables protective immunity even against tumours with common escape mutations.

Immunosuppressive Myeloid Cells Induce Nitric Oxide-Dependent DNA Damage and p53 Pathway Activation in CD8+ T Cells.

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.

A facile approach to enhance antigen response for personalized cancer vaccination.

Existing strategies to enhance peptide immunogenicity for cancer vaccination generally require direct peptide alteration, which, beyond practical issues, may impact peptide presentation and result in vaccine variability. Here, we report a simple adsorption approach using polyethyleneimine (PEI) in a mesoporous silica microrod (MSR) vaccine to enhance antigen immunogenicity. The MSR-PEI vaccine significantly enhanced host dendritic cell activation and T-cell response over the existing MSR vaccine and bolus vaccine formulations. Impressively, a single injection of the MSR-PEI vaccine using an E7 peptide completely eradicated large, established TC-1 tumours in about 80% of mice and generated immunological memory. When immunized with a pool of B16F10 or CT26 neoantigens, the MSR-PEI vaccine eradicated established lung metastases, controlled tumour growth and synergized with anti-CTLA4 therapy. Our findings from three independent tumour models suggest that the MSR-PEI vaccine approach may serve as a facile and powerful multi-antigen platform to enable robust personalized cancer vaccination.

Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell-driven tumor immunity.

MICA and MICB are expressed by many human cancers as a result of cellular stress, and can tag cells for elimination by cytotoxic lymphocytes through natural killer group 2D (NKG2D) receptor activation. However, tumors evade this immune recognition pathway through proteolytic shedding of MICA and MICB proteins. We rationally designed antibodies targeting the MICA α3 domain, the site of proteolytic shedding, and found that these antibodies prevented loss of cell surface MICA and MICB by human cancer cells. These antibodies inhibited tumor growth in multiple fully immunocompetent mouse models and reduced human melanoma metastases in a humanized mouse model. Antitumor immunity was mediated mainly by natural killer (NK) cells through activation of NKG2D and CD16 Fc receptors. This approach prevents the loss of important immunostimulatory ligands by human cancers and reactivates antitumor immunity.

A Micropolymorphism Altering the Residue Triad 97/114/156 Determines the Relative Levels of Tapasin Independence and Distinct Peptide Profiles for HLA-A(*)24 Allotypes.

While many HLA class I molecules interact directly with the peptide loading complex (PLC) for conventional loading of peptides certain class I molecules are able to present peptides in a way that circumvents the PLC components. We investigated micropolymorphisms at position 156 of HLA-A(*)24 allotypes and their effects on PLC dependence for assembly and peptide binding specificities. HLA-A(*)24:06(156Trp) and HLA-A(*)24:13(156Leu) showed high levels of cell surface expression while HLA-A(*)24:02(156Gln) was expressed at low levels in tapasin deficient cells. Peptides presented by these allelic variants showed distinct differences in features and repertoire. Immunoprecipitation experiments demonstrated all the HLA-A(*)24/156 variants to associate at similar levels with tapasin when present. Structurally, HLA-A(*)24:02 contains the residue triad Met97/His114/Gln156 and a Trp156 or Leu156 polymorphism provides tapasin independence by stabilizing these triad residues, thus generating an energetically stable and a more peptide receptive environment. Micropolymorphisms at position 156 can influence the generic peptide loading pathway for HLA-A(*)24 by altering their tapasin dependence for peptide selection. The trade-off for this tapasin independence could be the presentation of unusual ligands by these alleles, imposing significant risk following hematopoietic stem cell transplantation (HSCT).

Neuropathology in mouse models of mucopolysaccharidosis type I, IIIA and IIIB.

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.

Position 45 influences the peptide binding motif of HLA-B*44:08.

Position 45 represents a highly polymorphic residue within HLA class I alleles, which contacts the p2 position of bound peptides in 85% of the peptide-HLA structures analyzed, while the neighboring residues 41 and 46 are not involved in peptide binding. To investigate the influence of residue 45 at the functional level, we sequenced peptides eluted from recombinant HLA-B*44:08(41Ala/45Met/46Ala) molecules and compared their features with known peptides from B*44:02(41Thr/45Lys/46Glu). While HLA-B*44:02 has an anchor motif of E at the p2 anchor position, HLA-B*44:08 exhibits Q and L as anchor motif. The 45(Met/Lys) polymorphism contributes to the alteration in the peptide-binding motif and provides further evidence that mismatches at position 45 should be considered as nonpermissive in a transplantation setting.

Position 156 influences the peptide repertoire and tapasin dependency of human leukocyte antigen B*44 allotypes.

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.

Mismatches outside exons 2 and 3 do not alter the peptide motif of the allele group B*44:02P.

Sequence variations outside exons 2 and 3 do not appear to affect the function of human leukocyte antigen (HLA) class I alleles. HLA-B*44:02:01:01 and -B*44:27 are considered functionally identical because they differ by a single amino acid substitution of Val > Ala at position 199, which is located in the α3 domain. To validate that HLA-B*44:02:01:01 and -B*44:27 represent functionally identical alleles that might reflect a permissive mismatch in hematopoetic stem cell transplantation (HSCT), we determined their peptide-binding features. B-lymphoblastic cells were lentivirally transduced with B*44:02 and B*44:27 constructs and soluble recombinant molecules were purified by affinity chromatography. Peptides were isolated and sequencing of single peptides was performed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LTQ-Orbitrap) technology. We demonstrate that the peptide motif of B*44:02(199Val) and B*44:27(199Ala) is identical. Both variants feature E at P2 and Y, F, or W at PΩ in their ligands. Most of the identified peptides are 9 to 11 amino acids in length and approximately 20% of these ligands are shared between the alleles. Our results lead to the conclusion that B*44:02:01:01 and B*44:27 might have the same immune function, validating a theory that is now being used in deciding which donors to select in HSCT when there is no identical donor available.