Identification of Tumor Antigens in the HLA Peptidome of Patient-derived Xenograft Tumors in Mouse

1. Download : Download high-res image (147KB)
2. Download : Download full-size image

Highlights

• •Sufficient tumor tissues are often unavailable large HLA peptidome discovery.
• •Using patient derived xenograft (PDX) tumors can overcome this limitation.
• •The large PDX HLA peptidomes expand significantly those of the original biopsies.
• •The HLA peptidomes of the PDX tumors included many tumor antigens.

     

Lymph node colonization induces tumor-immune tolerance to promote distant metastasis

1. Download : Download high-res image (252KB)
2. Download : Download full-size image

     

Salicylic acid influences the protease activity and posttranslation modifications of the secreted peptides in the moss Physcomitrella patens

Plant secretome comprises dozens of secreted proteins. However, little is known about the composition of the whole secreted peptide pools and the proteases responsible for the generation of the peptide pools. The majority of studies focus on target detection and characterization of specific plant peptide hormones. In this study, we performed a comprehensive analysis of the whole extracellular peptidome, using moss Physcomitrella patens as a model. Hundreds of modified and unmodified endogenous peptides that originated from functional and nonfunctional protein precursors were identified. The plant proteases responsible for shaping the pool of endogenous peptides were predicted. Salicylic acid (SA) influenced peptide production in the secretome. The proteasome activity was altered upon SA treatment, thereby influencing the composition of the peptide pools. These results shed more light on the role of proteases and posttranslational modification in the “active management” of the extracellular peptide pool in response to stress conditions. It also identifies a list of potential peptide hormones in the moss secretome for further analysis.     

The Cbl-interacting protein TULA inhibits dynamin-dependent endocytosis

The Cbl- and ubiquitin-interacting protein T-cell ubiquitin ligand (TULA) has been demonstrated to inhibit endocytosis and downregulation of ligand-activated EGF receptor (EGFR) by impairing Cbl-induced ubiquitination. We presently report that TULA additionally inhibited clathrin-dependent endocytosis in general, as both uptake of transferrin (Tf) and low-density lipoprotein (LDL) was inhibited. Additionally, endocytosis of the raft proteins CD59 and major histocompatibility complex class I (MHC-I), which we demonstrate were mainly endocytosed clathrin-independently, but dynamin-dependently, was blocked in cells overexpressing TULA. By contrast, the uptake of ricin, which is mainly endocytosed clathrin- and dynamin-independently, was not affected by overexpressed TULA. Consistently, TULA and dynamin co-immunoprecipitated and colocalized intracellularly, and upon overexpression of dynamin the TULA-mediated inhibitory effect on endocytosis of Tf, LDL, CD59 and MHC-I was counteracted. Overexpressed dynamin did not restore ubiquitination of the EGFR, and consistently dynamin did not rescue endocytosis of the EGFR in cells overexpressing TULA. We conclude that TULA inhibits both clathrin-dependent and clathrin-independent endocytic pathways by functionally sequestering dynamin via the SH3 domain of TULA binding proline-rich sequences in dynamin.     

MALDI-TOF/TOF和LC-MS/MS液质联用分析大鼠脑脉络丛的多肽组

脉络丛(choroid plexus,CP)位于血液与脑脊液(cerebrospinal fluid,CSF)之间,不仅是CSF的重要来源,而且是构成血液-脑脊液屏障(blood-cerebrospinal fluid barrier,BCB)的组织基础.CP参与脑组织中一些血源性多肽的输送以及自身多肽合成的生理过程,在维持脑微环境动态平衡和调节中枢神经系统的正常功能方面起到非常重要的作用.本研究分别运用MALDI-TOF/TOF和LC-MS/MS液质联用系统分析了成年SD大鼠血液-脑脊液屏障(即脉络丛组织)中的多肽组.共鉴定到163个多肽(P0.001),这些多肽为69种蛋白质的降解肽段,其中ATP合酶(ATP synthase),细胞色素c(cytochrome c),血红蛋白(hemoglobin),NADH-辅酶Q氧化还原酶(NADH-ubiquinone oxidoreductase),β珠蛋白(beta-globin)这5种蛋白质的肽段数占总肽段数的50%以上,并且部分多肽序列相似度高,类似其前体蛋白的逐步降解片段,而这些前体蛋白质的分子量多数在10kD至20kD之间.上述研究结果为SD大鼠脉络丛组织的生理功能研究及组织多肽组学的研究方法提供了有价值的科学资料.     

DC-expressed MHC class I single-chain trimer-based vaccines prime cytotoxic T lymphocytes against exogenous but not endogenous antigens

The poor immunogenicity of many tumors can be partly explained by the inefficiency of the MHC class I peptide presentation pathway. MHC-I-based single-chain trimers (SCT) represent a new class of molecules with the potential to overcome this limitation. We here evaluated the ability of SCT presenting a melanoma antigen peptide (TRP-2) to prime cytotoxic T lymphocyte (CTL) responses in mice when given as DNA vaccines via Gene Gun or when expressed by dendritic cells. The SCT was unable to induce detectable priming or significant anti-tumor activity of CTL using either vaccination strategy, whereas control SCT (with an exogenous peptide) primed strong responses. This study thus provides the first data related to the use of SCT in combination with DC and their application toward self antigens and suggest this potent technology, alone, is insufficient to overcome self tolerance.     

The unfolded protein response in hereditary haemochromatosis

To cope with the accumulation of unfolded or misfolded proteins the endoplasmic reticulum (ER) has evolved specific signalling pathways collectively called the unfolded protein response (UPR). Elucidation of the mechanisms governing ER stress signalling has linked this response to the regulation of diverse physiologic processes as well as to the progression of a number of diseases. Interest in hereditary haemochromatosis (HH) has focused on the study of proteins implicated in iron homeostasis and on the identification of new alleles related with the disease. HFE has been amongst the preferred targets of interest, since the discovery that its C282Y mutation was associated with HH. However, the discrepancies between the disease penetrance and the frequency of this mutation have raised the possibility that its contribution to disease progression might go beyond the mere involvement in regulation of cellular iron uptake. Recent findings revealed that activation of the UPR is a feature of HH and that this stress response may be involved in the genesis of immunological anomalies associated with the disease. This review addresses the connection of the UPR with HH, including its role in MHC-I antigen presentation pathway and possible implications for new clinical approaches to HH.     

Immune Checkpoint Blockade Augments Changes Within Oncolytic Virus-induced Cancer MHC-I Peptidome,Creating Novel Antitumor CD8 T Cell Reactivities

The combination cancer immunotherapies with oncolytic virus (OV) and immune checkpoint blockade (ICB) reinstate otherwise dysfunctional antitumor CD8 T cell responses. One major mechanism that aids such reinstatement of antitumor CD8 T cells involves the availability of new class I major histocompatibility complex (MHC-I)-bound tumor epitopes following therapeutic intervention. Thus, therapy-induced changes within the MHC-I peptidome hold the key to understanding the clinical implications for therapy-reinstated CD8 T cell responses. Here, using mass spectrometry–based immuno-affinity methods and tumor-bearing animals treated with OV and ICB (alone or in combination), we captured the therapy-induced alterations within the tumor MHC-I peptidome, which were then tested for their CD8 T cell response-stimulating activity. We found that the oncolytic reovirus monotherapy drives up- as well as downexpression of tumor MHC-I peptides in a cancer type and oncolysis susceptibility dependent manner. Interestingly, the combination of reovirus + ICB results in higher numbers of differentially expressed MHC-I-associated peptides (DEMHCPs) relative to either monotherapies. Most importantly, OV+ICB-driven DEMHCPs contain biologically active epitopes that stimulate interferon-gamma responses in cognate CD8 T cells, which may mediate clinically desired antitumor attack and cancer immunoediting. These findings highlight that the therapy-induced changes to the MHC-I peptidome contribute toward the reinstated antitumor CD8 T cell attack established following OV + ICB combination cancer immunotherapy.     

Mass-Spectrometry Based Characterisation of Infant Whole Saliva Peptidome

The objective of the study was to determine optimal conditions for sampling, sample processing and mass-spectrometry based analysis of infants’ salivary peptidome. Saliva was sampled in 3- and 6-month-old infants and peptide extracts were prepared. Various sample pretreatments before profiling by MALDI-ToF were evaluated and peptide identification was undertaken by MALDI-ToF/ToF or nanoLC-ESI-IT tandem MS. A fast and simple protocol (cut-off filtration at 5 kDa) was satisfactory to produce extracts where no proteolysis was detected even when no protease inhibitor was added. Optimal MALDI spectra were generated after purification on C18 tips. Variability of spectra between two samples exceeded that of the technical replicates, validating that the method is suitable to conduct differential studies. Salivary peptides, identified by means of the two complementary mass spectrometry techniques, were fragments of proline-rich proteins and histatins. The fragments originated mainly from the C-terminal protein extremities. Indications on the proteolytic systems involved and the anatomic location where they intervene are proposed.     

Deciphering the MHC-associated peptidome: a review of naturally processed ligand data

Introduction: The availability of big data sets (‘OMICS’) has greatly impacted fundamental and translational science. High-throughput analysis of HLA class I and II associated peptidomes by mass spectrometry (MS) has generated large datasets, with the last decade witnessing tremendous growth in the breadth and number of studies.

Areas covered: For this, we first analyzed naturally processed peptide (NP) data captured within the IEDB to survey and characterize the current state of NP data. We next asked to what extent the NP data overlap with existing T cell epitope and MHC binding data.

Expert commentary: The current collection of NP data represents a large and diverse set of class I/II peptides mostly derived from self-antigens. These data overlap only marginally with existing immunogenicity and binding data and it is thus difficult to ascertain the correspondence between the different assay methodologies. This highlights a need for unbiased studies benchmarking in model antigen systems how well MHC binding and NP data predicts immunogenicity. Going forward, efforts at generating an integrated process for capturing all NP, curating associated metadata and accessing NP data from an immunological viewpoint will be important for development of novel methods for identifying optimal target antigens and for class I and II epitope prediction.