Dendritic cells (DCs) and B cells present antigen-derived peptides bound to MHC class II (MHC II) molecules for recognition by CD4-positive T lymphocytes. DCs control the intracellular traffic of peptide-MHC II complexes by regulating the ubiquitination of MHC II. In resting or "immature" DCs, ubiquitinated MHC II molecules are targeted to lysosomes, but upon pathogen-induced "maturation," ubiquitination is down-regulated and MHC II can accumulate on the plasma membrane of mature DCs. Although B cells constitutively ubiquitinate their MHC II, it unexpectedly remains at the surface. We find that DCs and B cells differ in MHC II-conjugated ubiquitin (Ub) chain length: four to six Ub in immature DCs vs. two to three in B cells. In both cell types, experimentally increasing Ub chain length led to efficient lysosomal transport of MHC II, whereas MHC II with fewer than two Ubs did not reach lysosomes. Thus, Ub chain length plays a crucial role in regulating the intracellular fate and function of MHC II in DCs and B cells.D endritic cells (DCs) and B lymphocytes are professional antigen-presenting cells (APCs) capable of stimulating efficient T-cell responses (1, 2). However, their approaches to antigen presentation differ in important respects. Whereas DCs are highly endocytic and internalize a wide variety of antigens, B cells take up and process only the single antigen recognized by their B-cell receptor. DCs are also distinguished by their ability to regulate antigen processing and presentation by "maturation" (3, 4). Immature DCs, found in peripheral tissues, are adept at endocytic uptake of antigen but do not efficiently generate peptide-MHC class II (MHC II) complexes or express them stably on the cell surface. In part, this is because MHC II in immature DCs is ubiquitinated on a single conserved lysine in the cytoplasmic domain of the β-chain (5, 6) by E3 ligases of the membrane-associated RING-CH (MARCH) family (7,8). Like other ubiquitinated membrane proteins (9), ubiquitinated MHC II molecules are targeted to and sequestered in multivesicular late endosomes and lysosomes. Upon receiving a maturation stimulus (e.g., Toll-like receptor agonist), however, ubiquitination ceases (5, 6) and peptide-MHC II complexes are translocated to and accumulate at the plasma membrane (10-13). In B cells, MHC II surface expression is always high despite also being ubiquitinated by MARCH ligases in naïve B cells (8).Internalization and down-regulation of receptor tyrosine kinases by ubiquitination is well known. Ligand binding activates the kinase, resulting in autophosophorylation and subsequent recruitment of soluble E3 ligases (e.g., Cbl) that ubiquitinate one or more acceptor lysines. The ubiquitin (Ub) moieties are recognized by Ub-interaction motif (UIM)-containing adapter molecules (e.g., epsins, eps15) that associate with clathrin-coated pits, leading to receptor internalization (14-18). Upon delivery to early endosomes, Ub is recognized by members of the endosomal sorting complex required for transport (ESCRT) complexe...

Studies in myeloid neoplasms have described recurrent IDH1 and IDH2 mutations as primarily mutually exclusive. Over a 6-month period of clinical testing with a targeted next-generation sequencing assay, we evaluated 92 patients with acute myeloid leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia and identified a subset of 21 patients (23%) who harbored mutations in either IDH1 or IDH2. Of the 21 patients with IDH mutations, 4 (19%) were found to have single nucleotide variants in both IDH1 and IDH2. An additional patient included in the study was found to have two different IDH2 mutations. The mutations were typically present at different variant allelic frequencies, with one predominating over the other, consistent with the presence of multiple subclones in a single patient. In one case, the variant allelic frequencies in both IDH1 and IDH2 were equally low in the setting of a high percentage of blasts, suggesting that the IDH mutations were unlikely to be present in the founding clone. Given these data, we conclude that dual IDH1/2 mutations likely were previously underestimated, a finding that may carry important treatment implications. (J Mol Diagn 2015, 17: 661e668; http://dx.doi.org/10.1016/j.jmoldx.2015 In recent years, whole-genome sequencing of acute myeloid leukemias (AMLs) led to the identification of frequent heterozygous mutations in isocitrate dehydrogenase 1 gene (IDH1).1,2 Subsequent exome sequencing and targeted resequencing studies in AML also identified frequent IDH2 mutations. 3,4 Overall, approximately 15% to 30% of AML have mutations in IDH1 or IDH2, almost exclusively at codon Arg132 for IDH1 and at codons Arg140 and Arg172 for IDH2.1,3e6 Patients with cytogenetically normal AML are enriched for these mutations. The mutations confer neomorphic enzyme activity through the NADPH-dependent reduction of the normal end-product a-ketoglutarate to the putative oncometabolite 2-hydroxyglutarate. The accumulation of high levels of 2-hydroxyglutarate in the IDH1/2-mutant tumor provides an important mechanism of cellular transformation through the targeting of epigenetic regulators. 7IDH mutations were also identified in preleukemic clonal malignancies, including myelodysplastic syndromes (MDSs) and myeloproliferative neoplasms (approximately 5% of MDS/myeloproliferative neoplasm and approximately 20% of AML arising from MDS or myeloproliferative neoplasm).

Background: B-cell lymphomas with concurrent translocations of MYC and BCL2 or BCL6, also known as ''double-hit'' lymphomas (DHL), are rare malignancies characterized by aggressive clinical behavior and poor prognosis. Previous reports suggest that decreased CD20 and/or CD19 expression by flow cytometry is relatively common in DHL and may help to identify cases requiring additional cytogenetic analysis.Methods: We conducted a retrospective analysis of 26 cases of DHL, and compared their flow cytometric characteristics to cases of Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). Cases were analyzed by four-color flow cytometry, and bivariate dot-plots were reviewed for light scatter characteristics, CD19, CD20, CD45, and surface light chain.Results: Relatively few DHL cases showed dim expression of CD19 or CD20, and statistically significant differences were found only in the frequency of dim CD19 expression between DHL and BL or DLBCL. Although concomitant dim CD19 and CD20 expression was exclusive to DHL, it was present in only a minority of cases.Conclusions: We conclude that although a subset of DHL expresses aberrant levels of CD19 and/or CD20 by flow cytometry, these findings are of limited utility in identifying cases requiring cytogenetic analysis due to their low frequency. Until more sensitive pathologic parameters can be identified and validated, the decision to perform cytogenetic analysis should rest on a combination of clinical, morphologic, and immunophenotypic features suggestive of high-grade, aggressive disease. V C 2013 International Clinical Cytometry Society