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The contribution of specific HLA Class II alleles in type 1 diabetes is determined by polymorphic amino acid epitopes that direct antigen binding therefore, along with conventional allele frequency analysis, epitope analysis can provide important insights into disease susceptibility. We analyzed the highly heterogeneous Cypriot population for the HLA class II loci of T1DM patients and controls and we report for the first time their allele frequencies. Within our patient cohort we identified a subgroup that did not carry the DRB1*03:01-DQA1*05:01-DQB1*02:01 and DRB1*04:xx-DQA1*03:01-DQB1*03:02 risk haplotypes but a novel recombinant one, DRB1*04:XX-DQA1*03:01-DQB1*02:01 designated DR4-DQ2.3. Through epitope analysis we identified established susceptibility (DQB1 A57, DRB1 H13) and resistance (DQB1 D57) residues as well as other novel susceptibility residues DRB1 Q70, DQB1 L26 and resistance residues DRB1 D70, R70 and DQB1 Y47. Prevalence of susceptibility epitopes was higher in patients and was not exclusively a result of linkage disequilibrium. Residues DRB1 Q70, DQB1 L26 and A57 and a 10 amino acid epitope of DQA1 were the most significant in discriminating risk alleles. An extended haplotype containing these epitopes was carried by 92% of our patient cohort. Sharing of susceptibility epitopes could also explain the absence of risk haplotypes in patients. Finally, many significantly associated epitopes were non-pocket residues suggesting that critical immune functions may exist spanning further from the binding pockets.

Competing Interests: Conflict-of-interest disclosure: G.S.V. is a consultant for KYMAB and receives an educational grant from Celgene. The remaining authors declare no competing financial interests.

Background: Major barriers in using classical FOXP3+ regulatory T cells (Tregs) in clinical practice are their low numbers in the circulation, the lack of specific cell surface markers for efficient purification and the loss of expression of Treg signature molecules and suppressive function after in vitro expansion or in a pro-inflammatory microenviroment. A surface molecule with potent immunosuppressive function is the human leukocyte antigen-G (HLA-G), which is normally expressed in placenta protecting the "semi-allogeneic" fetus from maternal immune attack. Because HLA-G expression is strongly regulated by methylation, we asked whether hypomethylating agents (HA) may be used in vitro to induce HLA-G expression on conventional T cells and convert them to Tregs., Methods: Human peripheral blood T cells were exposed to azacytidine/decitabine and analyzed for HLA-G expression and their in vitro suppressor properties., Results: HA treatment induces de novo expression of HLA-G on T cells through hypomethylation of the HLA-G proximal promoter. The HA-induced CD4 + HLA-G pos T cells are FOXP3 negative and have potent in vitro suppression function, which is dependent to a large extent, but not exclusively, on the HLA-G molecule. Converted HLA-G pos suppressors retain their suppressor function in the presence of tumor necrosis factor (TNF) and preserve hypomethylated the HLA-G promoter for at least 2 days after azacytidine exposure. Decitabine-treated T cells suppressed ex vivo the proliferation of T cells isolated from patients suffering from graft-versus-host disease (GVHD)., Discussion: We propose, in vitro generation of HLA-G-expressing T cells through pharmacological hypomethylation as a simple, Good Manufacturing Practice (GMP)-compatible and efficient strategy to produce a stable Treg subset of a defined phenotype that can be easily purified for adoptive immunotherapy., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

The strongest predictor of relapse in B-cell acute lymphoblastic leukemia (B-ALL) is the level of persistence of tumor cells after initial therapy. The high mutation rate of the B-cell receptor (BCR) locus allows high-resolution tracking of the architecture, evolution and clonal dynamics of B-ALL. Using longitudinal BCR repertoire sequencing, we find that the BCR undergoes an unexpectedly high level of clonal diversification in B-ALL cells through both somatic hypermutation and secondary rearrangements, which can be used for tracking the subclonal composition of the disease and detect minimal residual disease with unprecedented sensitivity. We go on to investigate clonal dynamics of B-ALL using BCR phylogenetic analyses of paired diagnosis-relapse samples and find that large numbers of small leukemic subclones present at diagnosis re-emerge at relapse alongside a dominant clone. Our findings suggest that in all informative relapsed patients, the survival of large numbers of clonogenic cells beyond initial chemotherapy is a surrogate for inherent partial chemoresistance or inadequate therapy, providing an increased opportunity for subsequent emergence of fully resistant clones. These results frame early cytoreduction as an important determinant of long-term outcome.

Acidification or glucocorticoids increase the maximal activity and subunit mRNA levels of branched chain alpha-ketoacid dehydrogenase (BCKAD) in various cell types. We examined whether these stimuli increase transcription of BCKAD subunit genes by transfecting BCKAD subunit promoter-luciferase plasmids containing the mouse E2 or human E1alpha-subunit promoter into LLC-PK(1) cells, which do not express glucocorticoid receptors, or LLC-PK(1)-GR101 cells, which we have engineered to constitutively express the glucocorticoid receptor gene. Dexamethasone or acidification increased luciferase activity in LLC-PK(1)-GR101 cells transfected with the E2 or E1alpha-minigenes; acidification augmented luciferase activity in LLC-PK(1) cells transfected with these minigenes but dexamethasone did not. A pH-responsive element in the E2 subunit promoter was mapped to a region >4.0 kb upstream of the transcription start site. Dexamethasone concurrently stimulated E2 subunit promoter activity and reduced the binding of nuclear factor-kappaB (NF-kappaB) to a site in the E2 promoter. Thus acidification and glucocorticoids independently enhance BCKAD subunit gene expression, and the glucocorticoid response in the E2 subunit involves interference with NF-kappaB, which may act as a transrepressor.

Regulation of the mammalian branched-chain alpha-ketoacid dehydrogenase complex (BCKAD) occurs under a variety of stressful conditions associated with changes in circulating glucocorticoids. Multiple levels of regulation in hepatocytes, including alteration of the levels of the structural subunits available for assembly (E1, alpha-ketoacid decarboxylase; E2, dihydrolipoamide acyltransferase; and E3, dihydrolipoamide dehydrogenase), as well as BCKAD kinase, which serves to phosphorylate the E1alpha subunit and inactivate complex activity, have been proposed. The direct role of glucocorticoids in regulating the expression of the murine gene encoding the major BCKAD subunit E2, upon which the other BCKAD subunits assemble, was therefore examined. Deletion analysis of the 5' proximal 7.0 kb of the murine E2 promoter sequence, using E2 promoter/luciferase expression minigene plasmids introduced into the hepatic H4IIEC3 cell line, suggested a promoter proximal region responsive to glucocorticoid regulation. Linker-scanning mutagenesis combined with deletion analysis established this functional glucocorticoid-responsive unit (GRU) to be located near the murine E2 proximal promoter site at -140 to -70 bp upstream from the transcription initiation site. The presence of this region in plasmid minigenes, containing varying amounts of the murine genomic sequence 5' upstream from proximal E2 promoter sequences, conferred 2-10 fold increases in luciferase reporter gene expression in H4IIEC3 cells, whether introduced by transient transfection or following co-selection for stable transfectants. The GRU region itself appeared to contain multiple interacting elements that combine to regulate overall E2 promoter activity in response to changing physiological conditions associated with varying concentrations of glucocorticoids and likely other hormonal effectors.

Alterations in dietary intake, especially of protein, may produce changes in the hepatic levels of the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex. The possible role of insulin in the regulation of these observed changes in hepatic capacity for BCKAD expression was therefore examined. Steady-state RNA levels encoding three of the subunits, E1 alpha, E1 beta and E2, increased by 2-4-fold in the livers of mice starved for 3 days, a known hypoinsulinaemic state. In contrast, the levels of E1 beta and E2, but not E1 alpha, RNA were decreased when mice were fed 0% protein diets compared with the levels observed in mice fed standard (23%) or higher protein isocaloric diets. BCKAD subunit protein levels under these conditions changed co-ordinately even though the changes in RNA were not co-ordinate. The effects of hormonal changes that might be associated with these dietary changes were examined, using the rodent hepatoma cell line H4IIEC3. In these cells, the levels of E1 alpha protein and mRNA were significantly depressed in the presence of insulin. In contrast, the levels of E1 beta and E2 RNAs were not decreased by insulin. The half-lives of the E1 alpha and E2 RNAs were determined to be quite long, from 13 to 18 h, with insulin having no dramatic overall effect on the half-lives determined over 24 h. Therefore, it is likely that insulin directly affects the transcription of the E1 alpha gene rather than RNA stability in exerting its negative regulatory effect. This effect is specific to the E1 alpha subunit. The differences in BCKAD subunit RNA levels observed under various nutritional and developmental conditions may therefore be the result of the differential effects of insulin and other hormones on the multiple regulatory mechanisms modulating BCKAD subunit expression.