Programmed −1 ribosomal frameshifting (−1 PRF) is a geneexpression mechanism used to express many viral and some cellular genes. In contrast, efficient natural utilization of −2 PRF has not been demonstrated previously in eukaryotic systems. Like all nidoviruses, members of the Arteriviridae (a family of positive-stranded RNA viruses) express their replicase polyproteins pp1a and pp1ab from two long ORFs (1a and 1b), where synthesis of pp1ab depends on −1 PRF. These polyproteins are posttranslationally cleaved into at least 13 functional nonstructural proteins. Here we report that porcine reproductive and respiratory syndrome virus (PRRSV), and apparently most other arteriviruses, use an additional PRF mechanism to access a conserved alternative ORF that overlaps the nsp2-encoding region of ORF1a in the +1 frame. We show here that this ORF is translated via −2 PRF at a conserved G_GUU_UUU sequence (underscores separate ORF1a codons) at an estimated efficiency of around 20%, yielding a transframe fusion (nsp2TF) with the N-terminal two thirds of nsp2. Expression of nsp2TF in PRRSVinfected cells was verified using specific Abs, and the site and direction of frameshifting were determined via mass spectrometric analysis of nsp2TF. Further, mutagenesis showed that the frameshift site and an unusual frameshift-stimulatory element (a conserved CCCANCUCC motif 11 nucleotides downstream) are required to direct efficient −2 PRF. Mutations preventing nsp2TF expression impair PRRSV replication and produce a small-plaque phenotype. Our findings demonstrate that −2 PRF is a functional gene-expression mechanism in eukaryotes and add another layer to the complexity of arterivirus genome expression.Nidovirales | virology | genetic recoding | overlapping gene | translation

JHFF and MG are inventors on a pending patent for the nonoptimized and optimized TRAV and TRBV sequences of TCRs for mutated NPM1 as well as cells containing these TCR sequences (no. 2019156).

Purpose: In human leukocyte antigen (HLA)–matched stem cell transplantation (SCT), it has been shown that beneficial immune response mediating graft-versus-tumor (GVT) responses can be separated from graft-versus-host disease (GVHD) immune responses. In this study, we investigated whether it would be possible to dissect the beneficial immune response of allo-HLA–reactive T cells with potent antitumor reactivity from GVHD-inducing T cells present in the detrimental immune response after HLA-mismatched SCT. Experimental Design: The presence of specific tumor-reactive T cells in the allo-HLA repertoire was analyzed at the time of severe GVHD after HLA-mismatched SCT, using tetramers composed of different tumor-associated antigens (TAA). Results: High-avidity allo-HLA-restricted T cells specific for the TAA preferentially expressed antigen on melanomas (PRAME) were identified that exerted highly single-peptide–specific reactivity. The T cells recognized multiple different tumor cell lines and leukemic cells, whereas no reactivity against a large panel of nonmalignant cells was observed. These T cells, however, also exerted low reactivity against mature dendritic cells (DC) and kidney epithelial cells, which was shown to be because of low PRAME expression. Conclusions: On the basis of potential beneficial specificity and high reactivity, the T-cell receptors of these PRAME-specific T cells may be effective tools for adoptive T-cell therapy. Clinical studies have to determine the significance of the reactivity observed against mature DCs and kidney epithelial cells. Clin Cancer Res; 17(17); 5615–25. ©2011 AACR.

The presented HLA class I ligands are the products of the intracellular processing machinery, with its continuous cycle of protein synthesis and degradation (3). Much is known about the proteins involved in antigen processing, but high fidelity ligand/epitope predictions are at present not possible. The discovery of additional involved enzymes (3, 4) and the exciting discovery of peptide splicing (5) have shown that antigen processing is even more complex than was previously thought. Moreover, gene expression studies have shown many nonstandard, unexpected protein products, including the production of antigens derived from aberrant protein fragments as a result of expression in alternative reading frames (6). Several studies report the identification of HLA ligands (7-10). Many results have been collected and discussed in a recent review on the large-scale analysis of HLA class I ligands (11). Collectively, these reports illustrate the need for in-depth elucidation of the HLA ligandome.Elucidation of T cell epitopes has traditionally been achieved with the use of a forward immunological approach, as pioneered by Hunt and coworkers (12,13). In this approach, the cognate peptide of T cells with the appropriate activity profile is elucidated via repeated rounds of chromatographic separation in combination with T cell recognition assays. Because T cells are not always available from the start, reverse immunological approaches (14 -17) have been developed to predict T cell epitopes through a combination of bioinformatics and in vitro proteasome digests. Predicted epitopes are synthesized and tested for their capability to activate T cells. The main disadvantage of this approach is that less than 0.1% of the peptides that survive intracellular processing are presented on HLA class I molecules (3).Therefore, we developed a large-scale peptidomics approach that is a reverse immunology approach based not on From the ‡Department of Immunohematology and Blood Transfusion,

MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here, we show that ectopic expression of miR-17, -20,-93 and -106, all AAAGUGC seedcontaining miRNAs, increases proliferation, colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1), an ubiquitinbinding protein and regulator of autophagy-mediated protein degradation, as a major target for these miRNAs in myeloid progenitors. In addition, we found increased expression of IntroductionMiRNAs are transcribed as long primary transcripts that are processed by RNaseIII endonucleases DROSHA and DICER into single-stranded RNAs of ϳ 22nt. 1 The nucleotides 2-7 at the 5Ј-end of miRNAs, referred to as the miRNA seed region, are important for miRNA target recognition. 2 MiRNAs regulate gene expression by pairing with the seed complementary sequences in the 3Ј untranslated region (UTR) of mRNAs. Most mammalian miRNAs both repress translation and enhance decay of their target transcript. 3,4 MiRNAs containing homologous seeds such as, for example, the Let-7 family of miRNAs, are believed to regulate the same targets. 2 Involvement of miRNAs in hematopoiesis is strongly suggested by the position of miRNA genes near translocation breakpoints and by their presence in loci targeted for deletion in human leukemias. 5 Furthermore, expression profiling data suggest a major role for miRNAs in regulation of hematopoietic cell commitment, proliferation, apoptosis, survival, and differentiation. [6][7][8][9] The importance of miRNAs during hematopoiesis has been shown by disruption of miRNA biogenesis in mice. For instance, Dicer-deleted hematopoietic stem cells are unable to reconstitute the hematopoietic system. 10 Further, conditional deletion of Dicer in T and B cells results in strong reduction of lymphocytes and diminished cell survival and functions. [11][12][13] Argonaute-2 knock-out in hematopoiesis results in impaired differentiation of B-lymphocytes and erythroid cells. 14,15 MiRNAs can be expressed in a cell type or tissue specific manner. For instance, miR-223 and miR-142 are almost exclusively expressed in hematopoietic cells. 16 MiR-223 is transcriptionally controlled by CCAAT/enhancer-binding protein ␣ (CEBPA) and suppresses the myeloid transcription factor MEF2C, a major regulator of progenitor cell proliferation and granulocyte specific functions. 17,18 In addition, specific miRNAs control cellular processes important for proliferation, survival, cytokine production and cell lineage decisions of developing T and B cells. 8,12 In hematopoietic stem cells, sustained expression of miR-155 causes a myeloproliferative disorder in mice. 19 Furthermore, forced miR-29a in hematopoietic precursors induces aberrant self-renewal and acute myeloid leukemia by still unidentified mechan...

On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated molecular information from formalin-fixed, paraffin-embedded (FFPE) tissue sections. In this work, we present the in situ multimodal analysis of N-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major analytical aspects, such as lateral diffusion of the analyte molecules and differences in measurement sensitivity due to the additional sample preparation methods, have been investigated for both N-glycans and proteolytic peptides. By combining the MSI approach with extract analysis, we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique N-glycan and peptide identities.

No abstract

Celiac disease is an enteropathy caused by intolerance to dietary gluten. The disorder is strongly associated with DQA1*0501/DQB1*0201 (HLA-DQ2) as ∼95% of celiac patients express this molecule. HLA-DQ2 has unique Ag-binding properties that allow it to present a diverse set of gluten peptides to gluten-reactive CD4+ T cells so instigating an inflammatory reaction. Previous work has indicated that the presence of negatively charged amino acids within gluten peptides is required for specific binding. This, however, only partly explains the scale of the interaction. We have now characterized 432 natural ligands of HLA-DQ2 representing length variants of 155 distinct sequences. The sequences were aligned and the binding cores were inferred. Analysis of the amino acid distribution of these cores demonstrated that negatively charged residues in HLA-DQ2-bound peptides are favored at virtually all positions. This contrasts with a more restricted presence of such amino acids in T cell epitopes from gluten. Yet, HLA-DQ2 was also found to display a strong preference for proline at several anchor and nonanchor positions that largely match the position of proline in gluten T cell epitopes. Consequently, the bias for proline at p6 and p8 facilitates the enzymatic conversion of glutamine into glutamic acid in gluten peptides at p4 and p6, two important anchor sites. These observations provide new insights in the unique ability of HLA-DQ2 to bind a large repertoire of glutamine- and proline-rich gluten peptides. This knowledge may be an important asset in the development of future treatment strategies.