Transmembrane Helices Are an Over-Presented and Evolutionarily Conserved Source of Major Histocompatibility Complex Class I and II Epitopes

Cytolytic T cell responses are predicted to be biased towards membrane proteins. The peptide-binding grooves of most alleles of histocompatibility complex class I (MHC-I) are relatively hydrophobic, therefore peptide fragments derived from human transmembrane helices (TMHs) are predicted to be presented more often as would be expected based on their abundance in the proteome. However, the physiological reason of why membrane proteins might be over-presented is unclear. In this study, we show that the predicted over-presentation of TMH-derived peptides is general, as it is predicted for bacteria and viruses and for both MHC-I and MHC-II, and confirmed by re-analysis of epitope databases. Moreover, we show that TMHs are evolutionarily more conserved, because single nucleotide polymorphisms (SNPs) are present relatively less frequently in TMH-coding chromosomal regions compared to regions coding for extracellular and cytoplasmic protein regions. Thus, our findings suggest that both cytolytic and helper T cells are more tuned to respond to membrane proteins, because these are evolutionary more conserved. We speculate that TMHs are less prone to mutations that enable pathogens to evade T cell responses.

MHC class I antigen cross-presentation mediated by PapMV nanoparticles in human antigen-presenting cells is dependent on autophagy

Nanoparticles made of the coat protein of papaya mosaic virus (PapMV) and a single-strand RNA were previously shown to be an efficient antigen presentation system for the trigger of cellular immunity. Engineering of PapMV nano with a cytotoxic T lymphocyte epitope was previously shown activating specific T lymphocytes through a proteasome-independent major histocompatibility complex class I (MHC-I) cross-presentation. In this study, we provide new insights into the mechanism of the MHC-I cross-presentation mediated by PapMV nanoparticles. We demonstrate that PapMV nanoparticles do not require the transporter associated with antigen presentation (TAP), but rather depend on lysosome acidification and cathepsin S protease activity for presentation of the T cell epitope. We have also linked the induction of autophagy with this vacuolar MHC-I cross-presentation process. Interestingly, autophagy is induced in antigen-presenting cells after PapMV nanoparticles exposure and inhibition of autophagy reduce MHC-I cross-presentation. This study demonstrates that autophagy is associated with TAP- and proteasome-independent MHC-I cross-presentation. A deeper understanding of the autophagy-dependent MHC-I cross-presentation will be useful in designing vaccination platforms that aim to trigger an efficient cytotoxic T lymphocyte response.

Glucocorticoid receptor regulates PD-L1 and MHC-I in pancreatic cancer cells to promote immune evasion and immunotherapy resistance

Despite unprecedented responses of some cancers to immune checkpoint blockade (ICB) therapies, the application of checkpoint inhibitors in pancreatic cancer has been unsuccessful. Glucocorticoids and glucocorticoid receptor (GR) signaling are long thought to suppress immunity by acting on immune cells. Here we demonstrate a previously undescribed tumor cell-intrinsic role for GR in activating PD-L1 expression and repressing the major histocompatibility complex class I (MHC-I) expression in pancreatic ductal adenocarcinoma (PDAC) cells through transcriptional regulation. In mouse models of PDAC, either tumor cell-specific depletion or pharmacologic inhibition of GR leads to PD-L1 downregulation and MHC-I upregulation in tumor cells, which in turn promotes the infiltration and activity of cytotoxic T cells, enhances anti-tumor immunity, and overcomes resistance to ICB therapy. In patients with PDAC, GR expression correlates with high PD-L1 expression, low MHC-I expression, and poor survival. Our results reveal GR signaling in cancer cells as a tumor-intrinsic mechanism of immunosuppression and suggest that therapeutic targeting of GR is a promising way to sensitize pancreatic cancer to immunotherapy.

Immunization with short peptide particles reveals a functional CD8+ T-cell neoepitope in a murine renal carcinoma model

BackgroundInduction of CD8+ T cells that recognize immunogenic, mutated protein fragments in the context of major histocompatibility class I (MHC-I) is a pressing challenge for cancer vaccine development.MethodsUsing the commonly used murine renal adenocarcinoma RENCA cancer model, MHC-I restricted neoepitopes are predicted following next-generation sequencing. Candidate neoepitopes are screened in mice using a potent cancer vaccine adjuvant system that converts short peptides into immunogenic nanoparticles. An identified functional neoepitope vaccine is then tested in various therapeutic experimental tumor settings.ResultsConversion of 20 short MHC-I restricted neoepitope candidates into immunogenic nanoparticles results in antitumor responses with multivalent vaccination. Only a single neoepitope candidate, Nesprin-2 L4492R (Nes2LR), induced functional responses but still did so when included within 20-plex or 60-plex particles. Immunization with the short Nes2LR neoepitope with the immunogenic particle-inducing vaccine adjuvant prevented tumor growth at doses multiple orders of magnitude less than with other vaccine adjuvants, which were ineffective. Nes2LR vaccination inhibited or eradicated disease in subcutaneous, experimental lung metastasis and orthotopic tumor models, synergizing with immune checkpoint blockade.ConclusionThese findings establish the feasibility of using short, MHC-I-restricted neoepitopes for straightforward immunization with multivalent or validated neoepitopes to induce cytotoxic CD8+ T cells. Furthermore, the Nes2LR neoepitope could be useful for preclinical studies involving renal cell carcinoma immunotherapy.

Proteomic Analysis Identifies FNDC1, A1BG, and Antigen Processing Proteins Associated with Tumor Heterogeneity and Malignancy in a Canine Model of Breast Cancer

New insights into the underlying biological processes of breast cancer are needed for the development of improved markers and treatments. The complex nature of mammary cancer in dogs makes it a great model to study cancer biology since they present a high degree of tumor heterogeneity. In search of disease-state biomarkers candidates, we applied proteomic mass spectrometry imaging in order to simultaneously detect histopathological and molecular alterations whilst preserving morphological integrity, comparing peptide expression between intratumor populations in distinct levels of differentiation. Peptides assigned to FNDC1, A1BG, and double-matching keratins 18 and 19 presented a higher intensity in poorly differentiated regions. In contrast, we observed a lower intensity of peptides matching calnexin, PDIA3, and HSPA5 in poorly differentiated cells, which enriched for protein folding in the endoplasmic reticulum and antigen processing, assembly, and loading of class I MHC. Over-representation of collagen metabolism, coagulation cascade, extracellular matrix components, cadherin-binding and cell adhesion pathways also distinguished cell populations. Finally, an independent validation showed FNDC1, A1BG, PDIA3, HSPA5, and calnexin as significant prognostic markers for human breast cancer patients. Thus, through a spatially correlated characterization of spontaneous carcinomas, we described key proteins which can be further validated as potential prognostic biomarkers.

LC3 and NLRC5 Interaction Inhibits NLRC5-Mediated MHC Class I Antigen Presentation Pathway in Endometrial Cancer

Background: The major histocompatibility complex class I (MHC- I) transactivator, nucleotide binding oligomerization domain-like receptor family caspase recruitment domain containing 5 (NLRC5), serves as a target for immune evasion in many cancers, including endometrial cancer (EC). An inhibition of autophagy can contribute to immunotherapy by assisting the MHC-I-mediated antigen presentation in cancer. However, the underlying mechanism for autophagy-regulated MHC-I in EC remains unclear. Our study aimed to investigate the effect of autophagy on NLRC5 and MHC-I-mediated antigen presentation, and to identify the potential mechanisms underlying this process in EC.Methods: We examined the levels of autophagy and MHC-I genes by performing transmission electron microscopy (TEM), RNA-seq sequencing, western blotting, and qRT-PCR. The t-test, F-test, Kaplan-Meier analysis, and Pearson’s correlation analysis were used for statistical evaluations of tissue microarrays. Immunofluorescence staining, co-immunoprecipitation (CO-IP), and glutathione S-transferase (GST) pull-down assay were performed. HEC-1A, AN3CA, and Ishikawa EC cells were transfected designed, and the role of LC3 and NLRC5 in MHC-I-mediated antigen presentation in EC was further evaluated in a xenotransplantation model of HEC-1A cell line. Results: Autophagy was upregulated in EC endometrium as compared to that in normal endometrium. MHC I and NLRC5 expressions were lower in EC endometrium than in normal endometrium. Autophagy played a negative role in the MHC-I genes expression in vitro. Furthermore, a negative correlation was found between LC3 and NLRC5 levels, and LC3 interacted with NLRC5 to inhibit NLRC5-mediated MHC-I antigen presentation pathway in vitro and in vivo. Conclusion: An upregulation of LC3 in EC patients may contribute to tumor immune escape by restricting the NLRC5-mediated MHC-I antigen presentation pathway, suggesting that inhibiting LC3 and promoting NLRC5 may be a promising immunotherapy strategy in the management of EC.

Integrative genomic and transcriptomic analysis in plasmablastic lymphoma identifies disruption of key regulatory pathways

Plasmablastic-lymphoma (PBL) represents a clinically heterogeneous subtype of aggressive-B-cell-non-Hodgkin-lymphoma. Although targeted-sequencing-studies and a single-center whole-exome-sequencing (WES) study in HIV+ patients recently revealed several genes, associated with PBL-pathogenesis, the global mutational-landscape and transcriptional-profile of PBL remain elusive. To inform on disease-associated mutational-drivers, mutational-patterns and perturbed pathways in HIV+ and HIV-PBL we performed WES and transcriptome sequencing (RNA-seq) of 33 PBL-tumors. Integrative analysis of somatic-mutations and gene-expression-profiles were performed to acquire insights into the divergent genotype-phenotype-correlation in EBV+ and EBV-PBL. We describe a significant accumulation of mutations in the Janus-kinase-signal-transducer and transcription-activator (OSMR, STAT3, PIM1, SOCS1) as well as receptor tyrosine-kinase RAS-pathways (ERBB3, NRAS, PDGFRB, NTRK). We provide further evidence of frequent perturbance of nuclear-factor κB (NFκB) signaling (NFKB2, BTK). Induced pathways, identified by RNA-seq closely resemble the mutational-profile regarding alterations accentuated in IL-6/JAK/STAT-signaling, NFκB-activity and MYC-signaling. Moreover, class I-MHC mediated antigen-processing and cell-cycle-regulation were significantly impacted by the EBV-status. An almost exclusive upregulation of PI3K/AKT/MTOR-signaling in EBV+ PBL and a significantly induced expression of NTRK3 in concert with recurrent oncogenic-mutations in EBV- PBL, hints at specific therapeutically targetable-mechanism in PBL-subgroups. Our characterization of a mutational and transcriptomic-landscape in PBL, distinct from DLBCL and MM substantiates the pathobiological-independence of PBL in the spectrum of B-cell-malignancies and thereby refines the taxonomy for aggressive-lymphomas.

Molecular and genomic characterisation of a panel of human anal cancer cell lines

Anal cancer is a rare disease that has doubled in incidence over the last four decades. Current treatment and survival of patients with this disease has not changed substantially over this period of time, due, in part, to a paucity of preclinical models to assess new therapeutic options. To address this hiatus, we set-out to establish, validate and characterise a panel of human anal squamous cell carcinoma (ASCC) cell lines by employing an explant technique using fresh human ASCC tumour tissue. The panel of five human ASCC cell lines were validated to confirm their origin, squamous features and tumourigenicity, followed by molecular and genomic (whole-exome sequencing) characterisation. This panel recapitulates the genetic and molecular characteristics previously described in ASCC including phosphoinositide-3-kinase (PI3K) mutations in three of the human papillomavirus (HPV) positive lines and TP53 mutations in the HPV negative line. The cell lines demonstrate the ability to form tumouroids and retain their tumourigenic potential upon xenotransplantation, with varied inducible expression of major histocompatibility complex class I (MHC class I) and Programmed cell death ligand 1 (PD-L1). We observed differential responses to standard chemotherapy, radiotherapy and a PI3K specific molecular targeted agent in vitro, which correlated with the clinical response of the patient tumours from which they were derived. We anticipate this novel panel of human ASCC cell lines will form a valuable resource for future studies into the biology and therapeutics of this rare disease.

Tight Regulation of Major Histocompatibility Complex I for the Spatial and Temporal Expression in the Hippocampal Neurons

The expression and function of immune molecules, such as major histocompatibility complex (MHC), within the developing and adult brain have been discovered over the past few years. Studies utilizing classical class I MHC knockout animals suggest that these molecules, in fact, play essential roles in the establishment, function, and modification of synapses in the CNS. Altered neuronal expression of class I MHC, as has been reported in pathological conditions, leads to aberrations in neuronal development and repair. In the hippocampus, cellular and molecular mechanisms that regulate synaptic plasticity have heretofore been extensively studied. It is for this reason that multiple studies directed at better understanding the expression, regulation, and function of class I MHC within the hippocampus have been undertaken. Since several previous reviews have addressed the roles of class I MHC in the formation and function of hippocampal connections, the present review will focus on describing the spatial and temporal expression of class I MHC in developing, healthy adult, and aging hippocampus. Herein, we also review current literatures exploring mechanisms that regulate class I MHC expression in murine hippocampus. With this review, we aim to facilitate a deeper mechanistic understanding into the complex tight regulation of MHC I expression in hippocampus, which are needed as we explore the potential for targeting MHC I for therapeutic intervention in normal aging and in neurodegenerative diseases in the future.

At the Cutting Edge against Cancer: A Perspective on Immunoproteasome and Immune Checkpoints Modulation as a Potential Therapeutic Intervention

Immunoproteasome is a noncanonical form of proteasome with enzymological properties optimized for the generation of antigenic peptides presented in complex with class I MHC molecules. This enzymatic property makes the modulation of its activity a promising area of research. Nevertheless, immunotherapy has emerged as a front-line treatment of advanced/metastatic tumors providing outstanding improvement of life expectancy, even though not all patients achieve a long-lasting clinical benefit. To enhance the efficacy of the currently available immunotherapies and enable the development of new strategies, a broader knowledge of the dynamics of antigen repertoire processing by cancer cells is needed. Therefore, a better understanding of the role of immunoproteasome in antigen processing and of the therapeutic implication of its modulation is mandatory. Studies on the potential crosstalk between proteasome modulators and immune checkpoint inhibitors could provide novel perspectives and an unexplored treatment option for a variety of cancers.