Human Lymphoid Cells Research Articles

Differential Interactions of Falcarinol Combined with Anti-Tumour Agents on Cellular Proliferation and Apoptosis in Human Lymphoid Leukaemia Cell Lines

Leukaemia is the most common childhood cancer, and whilst recent advances in therapy have improved survival, current treatments are still limited by their side effects. Thus, new therapies are urgently needed, this study investigated the effects of Falcarinol, a polyacetylene isolated from carrots (Daucus carota) in combination with chemotherapy agents, anti-cancer agents and other apoptosis inducers. Inhibition of cellular proliferation and induction of apoptosis were investigated in three human lymphoid Leukaemia cell lines. Cellular proliferation was determined via ATP quantification using the Cell Titer Glo assay. Induction of apoptosis was investigated using caspase 3 activity assay and confirmed by nuclear morphology using Hoechst 33342. The study demonstrated that CCRF-CEM cells failed to induce synergistic response with any of the investigated chemotherapies, but importantly no inhibition was observed either. Jurkat cells showed a significant synergistic induction of apoptosis following joint treatment with Falcarinol and a Death Receptor 5 agonist (DR5), whereas CCRF-CEM cells showed only an additive response. Conversely within MOLT-3 cells Falcarinol partially inhibited the induction of apoptosis by DR5 agonist although this failed to reach significance. However MOLT-3 cells demonstrated synergistic induction of apoptosis when Falcarinol was combined with either Bortezomib (proteosome inhibitor), or Sulforaphane (histone deacetylase inhibitor). Identification of interactions between natural bioactive compounds with anti-cancer drugs may provide new pathways to target cancerous cells. Furthermore, since some combinations enhance apoptosis but some inhibit apoptosis it may be important to consider these interactions for dietary advice during therapy.

Innate lymphocyte cells in asthma phenotypes.

T helper type 2 (TH2) cells were previously thought to be the main initiating effector cell type in asthma; however, exaggerated TH2 cell activities alone were insufficient to explain all aspects of asthma. Asthma is a heterogeneous syndrome comprising different phenotypes that are characterized by their different clinical features, treatment responses, and inflammation patterns. The most-studied subgroups of asthma include TH2-associated early-onset allergic asthma, late-onset persistent eosinophilic asthma, virus-induced asthma, obesity-related asthma, and neutrophilic asthma. The recent discovery of human innate lymphoid cells capable of rapidly producing large amounts of cytokines upon activation and the mouse data pointing to an essential role for these cells in asthma models have emphasized the important role of the innate immune system in asthma and have provided a new means of better understanding asthma mechanisms and differentiating its phenotypes.

Flow Cytometric Analysis of Human Small Intestinal Lymphoid Cells.

Flow cytometry is a powerful technique allowing simultaneous analysis of numerous morphological and phenotypic characteristics of cells and cellular constituents. Improvements in cell isolation techniques in recent years have enabled flow cytometric analyses of cells derived from tissue biopsies. Here we describe a method for isolating and analyzing small intestinal lymphoid cells using flow cytometry.

Synthesis and biological evaluation of furanoallocolchicinoids.

A series of conformationally flexible furan-derived allocolchicinoids was prepared from commercially available colchicine in good to excellent yields using a three-step reaction sequence. Cytotoxicity studies indicated the potent activity of two compounds against human epithelial and lymphoid cell lines (AsPC-1, HEK293, and Jurkat) as well as against Wnt-1 related murine epithelial cell line W1308. The results of in vitro experiments demonstrated that the major effect of these compounds was the induction of cell cycle arrest in the G2/M phase as a direct consequence of effective tubulin binding. In vivo testing of the most potent furanoallocolchicinoid 10c using C57BL/6 mice inoculated with Wnt-1 tumor cells indicated significant inhibition of the tumor growth.

GNB1 Activating Mutations Promote Myeloid and Lymphoid Neoplasms Targetable By Combined PI3K/mTOR Inhibition

GNB1 encodes a beta subunit (Gβ) of heterotrimeric G proteins, which mediate signals downstream of G protein coupled receptors (GPCRs). We isolated a somatic mutant of GNB1 (K89E) by functional screening of a cDNA library derived from a blastic plasmacytoid dendritic cell neoplasm (BPDCN). A search of cancer genome databases identified recurrent mutations in GNB1 and the highly related protein GNB2. GNB1/2 K89E/T were found in B cell acute lymphoblastic leukemia (B-ALL) (1 case), follicular lymphoma (1) and myelodysplastic syndrome (MDS) (1) as well as BPDCN (1). Interestingly GNB1 K57E/T mutations were found only in myeloid diseases: [acute myeloid leukemia (2), atypical CML (2), polycythemia vera (1) and MDS (6)], while GNB1 I80N/T were found predominantly in B cell diseases [CLL (2), FL (2), DLBCL (1) and MDS (1)]. These mutated codons are all located on the GNB1 protein surface that is critical for interactions between Gβ and alpha subunits (Gα) or downstream effectors. Immunoprecipitation followed by mass spectrometry demonstrated that GNB1 K57E, I80T and K89E mutants failed to bind Gα, including GNAI2/3, GNA11/Q and GNA13 that are normally bound by wild-type (WT) GNB1. All mutations affecting these codons promoted cytokine-independent growth of human TF1 myeloid cells or mouse BaF3 lymphoid cells with activation of MEK/ERK and mTOR/PI3K pathways. Pertussis toxin treatment did not affect GNB1-dependent ERK activation or cell growth, implying a Gα-independent pathway. To investigate the function of GNB1 mutations in vivo, we performed a mouse bone marrow transplantation (BMT) experiment using wild-type and Cdkn2a-deficient donors. Loss of the cell cycle regulator CDKN2A is common in BPDCN, B-ALL, and several other hematologic malignancies. Bone marrow cells were isolated from 5-FU treated donor mice and infected with retrovirus expressing GNB1 WT, K57E, I80T or K89E. Transplantation of GNB1 mutant-expressing Cdkn2a-deficient bone marrow resulted in myeloid dendritic cell neoplasms that were CD11b+, CD11c+, CD19-, B220-, and CD3-. GNB1 mutants did not induce tumors in WT bone marrow after 12 months of observation suggesting that GNB1 requires additional cooperating mutations such as Cdkn2a loss. We performed the same BMT experiment using Cdkn2a-deficient bone morrow cells without 5-FU pretreatment. We found thatGNB1 I80T and K89E mutants induced a progenitor B cell ALL (CD11b-, CD11c-, CD19+, CD3-, TdT+). These data suggest that GNB1 mutations can promote tumorigenesis in more than one cell lineage, as observed in patients. In vivo treatment of the myeloid neoplasm with the dual PI3K/mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, we noted that GNB1 mutations co-occurred with oncogenic kinase alterations, including BCR/ABL, JAK2 V617F and BRAF V600K. Co-expression of patient-derived GNB1 alleles with the mutant kinases resulted in relative resistance to treatment with the corresponding kinase inhibitor in each context. Thus, GNB1 and GNB2 mutations confer transformation and targeted therapy resistance across a range of human tumors and may be targetable with inhibitors of PI3K/mTOR signaling. DisclosuresGotlib:Novartis Pharmaceuticals Corporation: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Travel Support Other. Deininger:BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMS, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy. Tyner:Constellation Pharmaceuticals: Research Funding.

Minimizing inhibition of PCR-STR typing using digital agarose droplet microfluidics.

The presence of PCR inhibitors in forensic and other biological samples reduces the amplification efficiency, sometimes resulting in complete PCR failure. Here we demonstrate a high-performance digital agarose droplet microfluidics technique for single-cell and single-molecule forensic short tandem repeat (STR) typing of samples contaminated with high concentrations of PCR inhibitors. In our multifaceted strategy, the mitigation of inhibitory effects is achieved by the efficient removal of inhibitors from the porous agarose microgel droplets carrying the DNA template through washing and by the significant dilution of targets and remaining inhibitors to the stochastic limit within the ultralow nL volume droplet reactors. Compared to conventional tube-based bulk PCR, our technique shows enhanced (20 ×, 10 ×, and 16 ×) tolerance of urea, tannic acid, and humic acid, respectively, in STR typing of GM09948 human lymphoid cells. STR profiling of single cells is not affected by small soluble molecules like urea and tannic acid because of their effective elimination from the agarose droplets; however, higher molecular weight humic acid still partially inhibits single-cell PCR when the concentration is higher than 200 ng/μL. Nevertheless, the full STR profile of 9948 male genomic DNA contaminated with 500 ng/μL humic acid was generated by pooling and amplifying beads carrying single-molecule 9948 DNA PCR products in a single secondary reaction. This superior performance suggests that our digital agarose droplet microfluidics technology is a promising approach for analyzing low-abundance DNA targets in the presence of inhibitors.

Abstract 16: Immunomodulatory effects of VEGF on human lymph node antigen-presenting and lymphoid cells

Abstract Background: Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic signal associated with the growth and metastasis of tumors. In addition, VEGF has also been implicated in the suppression of immune cell function. The purpose of this study was to characterize VEGF secretion, receptor expression and effect of soluble VEGF neutralization in human lymph node cells both at rest and during activation. Methods: Human lymph nodes derived from patients with melanoma (IRB CASE 3610) containing T cells, B cells, monocyte/macrophages and dendritic cells were cultured with anti-CD3/CD28 beads and IL-2 in order to induce an inflammatory response in vitro. Culture supernatant was assayed for the presence of soluble VEGF on days 4, 7, 11 and 14 of culture. Multiparameter FACS analysis was performed to assess the cell surface expression of VEGF receptor subtypes 1 and 2 over the 14-day culture period. In addition, intracellular expression of cytokines within effector T cells was assessed at the end of the culture. Finally, a parallel culture containing a VEGF neutralizing antibody was performed to assess the effect of VEGF neutralization on effector T cell phenotype. High-throughput T cell receptor CDR3 sequencing was used to determine the molecular sequence of the variable region of the T cell receptors (ImmunoSEQ, Seattle, WA). Results: VEGF was measureable in the culture supernatant on days 4 and beyond. The proportion of cells expressing intracellular VEGF peaked at day 4 and subsequently decreased. CD11b+(monocyte/macrophage), CD11c+ (dendritic cells) and CD19+ (B) cells had the highest proportion of cells which expressed intracellular VEGF. VEGF R1 and R2 were not constitutively expressed on resting immune cells, but receptor expression was upregulated upon activation of the T cells in vitro. Day 14 T cells demonstrated intracellular IFN-γ, IL-2 and TNF-α. The addition of VEGF-blocking antibody in the culture of MDLN cells effectively neutralized the amount of soluble VEGF detected in the culture supernatant at all days. The addition of the VEGF-blocking antibody also increased the proportion of T cells expressing intracellular IFN-γ as compared T cells in the non-VEGF blocked culture and resulted in distinct molecularly defined T cell clones. Conclusion: These results confirm that soluble VEGF is produced by immune cells during activation, even in the absence of tumor. VEGF receptor expression on lymph node immune cell subsets is constitutive, but low, and then upregulated during in vitro activation of T cells, presumably due to secondary cytokine production. Neutralization of soluble VEGF resulted in qualitative differences in effector cytokine expression and T cell receptor sequences. Whether the effect on these T cells is direct via VEGF interaction with the cognate receptor or from an indirect effect on antigen-presenting cells is currently under investigation. Citation Format: Madeleine P. Strohl, Hallie Graor, Mei Zhang, Anthony Visioni, John Ammori, Isabelle Rivers-McCue, Julian Kim. Immunomodulatory effects of VEGF on human lymph node antigen-presenting and lymphoid cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 16. doi:10.1158/1538-7445.AM2014-16

High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris.

Single-chain variable fragments (scFvs) have recently emerged as attractive candidates in targeted immunotherapy of various malignancies. The anti-CD22 scFv is able to target CD22, on B cell surface and is being considered as a promising molecule in targeted immunotherapy of B cell malignancies. The recombinant anti-CD22 scFv has been successfully expressed in Escherichia coli; however, the insufficient production yield has been a major bottleneck for its therapeutic application. The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the production of a wide variety of recombinant proteins such as antibody fragments. In this study, we used the Pichia expression system to express a humanized scFv antibody against CD22. The full-length humanized scFv gene was codon optimized, cloned into the pPICZαA and expressed in GS115 strain. The maximum production level of the scFv (25 mg/L) were achieved at methanol concentration, 1 %; pH 6.0; inoculum density, OD600 = 3 and the induction time of 72 h. The correlation between scFv gene dosage and expression level was also investigated by real-time PCR, and the results confirmed the presence of such correlation up to five gene copies. Immunofluorescence and flow cytometry studies and Biacore analysis demonstrated binding to CD22 on the surface of human lymphoid cell line Raji and recombinant soluble CD22, respectively. Taken together, the presented data suggest that the Pichia pastoris can be considered as an efficient host for the large-scale production of anti-CD22 scFv as a promising carrier for targeted drug delivery in treatment of CD22(+) B cell malignancies.

PTU-119 Phenotypic Characteristics And Localisation Of Novel Human Liver Infiltrating Nkp46 Subsets

IntroductionCD56+Natural killer cells are the principal effector cells of the innate immune system and have a well-established role in tumour surveillance and anti-viral immunity. Expression of NKp46 has been shown...

Cytotoxic activity of ethanolic extracts of Eleutherococcus species cultivated in Poland on HL60 leukemia cell line

Abstract The Eleutherococcus species including 40 species native to Asia are medicinal plants widely used in traditional medicine. Some of these species are cultivated at the botanical gardens in Europe. On the basis on our earlier studies it was concluded that the extracts of analyzed species act as antioxidants, inhibitors of MMPs, and cytotoxic against Jurkat 45 leukemia cell line. In this study, the anti-leukemic potential of roots and leaves from six Eleutherococcus species cultivated in Poland was determined. The in vitro cytotoxic activity towards human promyelotic leukemia cell line HL60 using trypan blue assay was evaluated. The induction of apoptosis in stimulated leukemia cells was determined by AnnexinV method. Morphological changes in treated cells were observed by microscopic investigations. The results showed that ethanolic extracts from the roots and the leaves of E. senticosus, E. setchuensis, E. sessiliflorus, E.gracilistylus, E. henryi and E. divaricatus exhibit cytotoxic effect towards leukemic HL60 cells. The received IC50 values for roots ranged from 49- 208 μg/mL and for the leaves from 116-518 μg/mL. The ethanol extract from the roots of E. divaricatus showed the highest cytotoxic and proapoptotic effect on HL60 human lymphoid leukemia cell line.

Su1002 Pyrosequencing Analysis of the Synonymous Codon Usage Pattern of rtN236T Mutation in 1418 Cases of Chronic Hepatitis B Patients Treated With Adefovir Dipivoxil

[Background] We previously reported that soluble factors produced from hepatocyte could contribute to the immune pathogenesis of HBV-related inflammation and progression of HCC. (JID2010, JG2012). It has been reported that the progression of fibrosis and HCC is different among the patients infected with various HBV genotypes and HCV. [Aim] The aim of this study is to analyze the effects of chemokines derived from HBV (genotype A, B and C) and HCV -infected hepatocytes and hepatoma cells for the various kinds of immune cells in vitro and in vivo. [Methods] Plasmid Construction: At least 5 clones of each fragment were sequenced and consensus sequence was identified and used as template for 1.24-fold the HBV genome of different genotypes (2 strains each for A, B and C). Cells: Huh7 and HepG2 cells were used for the experiment of transfection. cDNA array: cDNA array for 84chemokine-related genes were carried out. Flow-cytometry analysis: The expression of NKG2D and NKG2A on the NK cells, PD1 on CD8+ cells and MHC class I-related molecule A (MICA) on the HCC cells were analyzed by FACS canto-II. Migration Assay: various kinds of human primary lymphocyte were negatively isolated by MACS beads and used for the migration assay. NOG Mouse model: NOG mice were used for HBV-expressed HCC-transplanted-mouse model. Human primary lymphoid cells were introduced into this mouse. Immunohistochemistry: The liver samples obtained from 20 HBV related HCC patients, HCV-related HCC patients and non HBV/HCV-related HCC patients were stained with CX3CL1, CX3CR1, CD3 and CD56 antibodies. [Results] CCBP2, IL18, CMTM3, CMTM4, CX3CL1, CXCL12, TYMP, GPR81, LTB4R, SDF2 and VHL were well expressed (threshold cycle value range<28) in HBV-replicated-Huh7 cells and significantly up-regulated in comparison to mock transfected Huh7 cells (p<0.01). Among these chemokines-related genes, the expression level of CX3CL1 mRNA was significantly different among genotype A, B and C (relative expression: 1.41, 2.04 and 3.31) (p<0.05). The migration activity of CD16+CD56dimNK cells, CD4+ cells, CD8+ cells and CD14+ Monocyte but not CD19+ B cells or Tregs cells were significantly different among genotype A, B and C in vitro (p<0.05). Moreover, the neutralization of CX3CL1 could cancel these differences of migration activities. Significantly lower expression of NKG2D on CX3CR1+NK cells were observed in primary human peripheral blood mononuclear cells obtained from HBV-related HCC patients and the Tumor-infiltrating lymphocytes in HCC with HBV expression in NOG mouse model in comparison to those is non-HBV related HCC. (p<0.05). [Conclusion] The differential expre s s ion l eve l s o f CX3CL1 and MICA and the d i f f e r en t i a l a t t r ac t i on of NKG2DlowCX3CR1+NK cells and PD-1+ CX3CR1+ CD8 T cells might be involved in HBV persistent infection and hepatocellular carcinoma by affecting immune cells.

Su1000 Hepatoma With HBV Replication Could Attract CX3CR1+ NKG2Dlow NK Cells and CX3CR1+PD-1+ CD8 T Cells

[Background] We previously reported that soluble factors produced from hepatocyte could contribute to the immune pathogenesis of HBV-related inflammation and progression of HCC. (JID2010, JG2012). It has been reported that the progression of fibrosis and HCC is different among the patients infected with various HBV genotypes and HCV. [Aim] The aim of this study is to analyze the effects of chemokines derived from HBV (genotype A, B and C) and HCV -infected hepatocytes and hepatoma cells for the various kinds of immune cells in vitro and in vivo. [Methods] Plasmid Construction: At least 5 clones of each fragment were sequenced and consensus sequence was identified and used as template for 1.24-fold the HBV genome of different genotypes (2 strains each for A, B and C). Cells: Huh7 and HepG2 cells were used for the experiment of transfection. cDNA array: cDNA array for 84chemokine-related genes were carried out. Flow-cytometry analysis: The expression of NKG2D and NKG2A on the NK cells, PD1 on CD8+ cells and MHC class I-related molecule A (MICA) on the HCC cells were analyzed by FACS canto-II. Migration Assay: various kinds of human primary lymphocyte were negatively isolated by MACS beads and used for the migration assay. NOG Mouse model: NOG mice were used for HBV-expressed HCC-transplanted-mouse model. Human primary lymphoid cells were introduced into this mouse. Immunohistochemistry: The liver samples obtained from 20 HBV related HCC patients, HCV-related HCC patients and non HBV/HCV-related HCC patients were stained with CX3CL1, CX3CR1, CD3 and CD56 antibodies. [Results] CCBP2, IL18, CMTM3, CMTM4, CX3CL1, CXCL12, TYMP, GPR81, LTB4R, SDF2 and VHL were well expressed (threshold cycle value range<28) in HBV-replicated-Huh7 cells and significantly up-regulated in comparison to mock transfected Huh7 cells (p<0.01). Among these chemokines-related genes, the expression level of CX3CL1 mRNA was significantly different among genotype A, B and C (relative expression: 1.41, 2.04 and 3.31) (p<0.05). The migration activity of CD16+CD56dimNK cells, CD4+ cells, CD8+ cells and CD14+ Monocyte but not CD19+ B cells or Tregs cells were significantly different among genotype A, B and C in vitro (p<0.05). Moreover, the neutralization of CX3CL1 could cancel these differences of migration activities. Significantly lower expression of NKG2D on CX3CR1+NK cells were observed in primary human peripheral blood mononuclear cells obtained from HBV-related HCC patients and the Tumor-infiltrating lymphocytes in HCC with HBV expression in NOG mouse model in comparison to those is non-HBV related HCC. (p<0.05). [Conclusion] The differential expre s s ion l eve l s o f CX3CL1 and MICA and the d i f f e r en t i a l a t t r ac t i on of NKG2DlowCX3CR1+NK cells and PD-1+ CX3CR1+ CD8 T cells might be involved in HBV persistent infection and hepatocellular carcinoma by affecting immune cells.

Epigenetic alteration by DNA-demethylating treatment restores apoptotic response to glucocorticoids in dexamethasone-resistant human malignant lymphoid cells.

BackgroundGlucocorticoids (GCs) are often included in the therapy of lymphoid malignancies because they kill several types of malignant lymphoid cells. GCs activate the glucocorticoid receptor (GR), to regulate a complex genetic network, culminating in apoptosis. Normal lymphoblasts and many lymphoid malignancies are sensitive to GC-driven apoptosis. Resistance to GCs can be a significant clinical problem, however, and correlates with resistance to several other major chemotherapeutic agents.MethodsWe analyzed the effect of treatment with the cytosine analogue 5 aza-2’ deoxycytidine (AZA) on GC resistance in two acute lymphoblastic leukemia (T or pre-T ALL) cell lines- CEM and Molt-4- and a (B-cell) myeloma cell line, RPMI 8226. Methods employed included tissue culture, flow cytometry, and assays for clonogenicity, cytosine extension, immunochemical identification of proteins, and gene transactivation. High throughput DNA sequencing was used to confirm DNA methylation status.ConclusionsTreatment of these cells with AZA resulted in altered DNA methylation and restored GC-evoked apoptosis in all 3 cell lines. In CEM cells the altered epigenetic state resulted in site-specific phosphorylation of the GR, increased GR potency, and GC-driven induction of the GR from promoters that lie in CpG islands. In RPMI 8226 cells, expression of relevant coregulators of GR function was altered. Activation of p38 mitogen-activated protein kinase (MAPK), which is central to a feed-forward mechanism of site-specific GR phosphorylation and ultimately, apoptosis, occurred in all 3 cell lines. These data show that in certain malignant hematologic B- and T-cell types, epigenetically controlled GC resistance can be reversed by cell exposure to a compound that causes DNA demethylation. The results encourage studies of application to in vivo systems, looking towards eventual clinical applications.

Apoptotic effect of synthetic 2′,4′,5′-trimethoxychalcones in human K562 and Jurkat leukemia cells

In this study, we assessed the cytotoxic effect of synthetic 2′,4′,5′-trimethoxychalcones on the human K562 acute myeloid leukemia cell and human Jurkat acute lymphoid leukemia cell. Compounds 13, 16, 19, and 26 showed low IC50 values (4.10–8.56 μM at 72 h) for both cell lines and did not have a cytotoxic effect on normal human lymphocytes. The mechanism of cell death induced by these compounds involves a decrease in the expression of cell proliferation marker Ki67, suggesting inhibition of cell proliferation. Furthermore, these chalcones reduced mitochondrial potential, decreased Bcl-2 expression, and increased Bax expression, indicating that the mechanism of apoptosis induced by them involves the intrinsic apoptosis pathway. The mechanism of action also involves increase in active caspase-3 and decrease in survivin expression. These results support the chalcones as potential antitumoral agents for further optimization.

Increased Diversity of the HLA-B40 Ligandome by the Presentation of Peptides Phosphorylated at Their Main Anchor Residue

Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I-associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy.

To B or Not to B? Glucocorticoid Impact on B Lymphocyte Fate and Function

Lymphocytes have been used to study the action of glucocorticoids for more than a half-century. Although it had been recognized that adrenal steroids administered in vivo could cause atrophy of the thymus and lymph nodes of the rat (1), Dr Allan Munck and his colleagues were the first to show that a specific biochemical change (reduction in glucose use) could be elicited by glucocorticoids acting on thymocytes and that these effects seem to be mediated through hormone interaction with highly specific intracellular binding sites (2–5). These receptors were also recognized to mediate the apoptotic response of lymphoid cells of some species, and murine lymphoma cell lines that exhibited resistance to these lytic effects of glucocorticoids were used as a tool to explore the structure-function relationships of the glucocorticoid receptor (GR) (6–9). The power of this approach drove many important discoveries in the field of steroid hormone action. In what has seemed at times like a parallel universe, glucocorticoids were being applied in the clinical setting for the control of both normal and abnormal immune system function long before we knew anything about how they worked (10). These hormonal compounds were used to treat patients with immune system neoplasms and autoimmune, allergic, or inflammatory diseases as well as those undergoing organ transplantation, even though the mechanisms by which these various clinical effects were mediated remained largely obscure. We have now begun to recognize that the glucocorticoid actions on the immune system likely extend beyond apoptotic responses and suppression of cytokine gene expression in thymus-derived lymphocytes (11). This recognition has led to the expectation that other specific cellular and molecular targets of glucocorticoids can be identified in the immune system and, hopefully, exploited for therapeutic application in the clinical setting. In this issue of Endocrinology, Gruver-Yates et al (12) present an exploration of the expression of the GR across the developmental pathway of (bone marrow derived) Blymphocytes, the antibody-producing cells of the humoral immune system. The authors use flow cytometric techniques to meticulously characterize GR expression in immature B cell subsets extracted from bone marrow and from peripheral lymphoid organs, and they demonstrate functionality of these receptors by assessing the apoptotic responses of these cells to glucocorticoids administered in vitro and in vivo. Their results are of particular interest in that they identify B-lymphocytes at all stages of development as potential targets of glucocorticoid action. Although endocrinologists seem to have heretofore largely ignored the B lymphocyte as a glucocorticoid target, immunologists (for at least a time) showed considerable interest in the idea. In vivo experiments in humans receiving supraphysiological doses of exogenous glucocorticoids [100 or 400 mg of hydrocortisone (13) or 60 mg of prednisone (14)] resulted in transient reductions in the number of circulating lymphocytes after 4–6 hours. These effects reverted to normal by 24 hours. The most pronounced reductions occurred in T cells, but circulating B cell numbers were also reduced by nearly 50% (14). The ability of the remaining circulating lymphocytes to respond to phytohemagglutinin (a T cell mitogen) was unaltered, but responses to pokeweed mitogen (a B cell mitogen) were reduced by the highest dose of hydrocortisone (13). Because previous studies had demonstrated resistance of human lymphoid cells to glucocorticoid-mediated lysis (15), the investigators interpreted their findings in

Li/Na exchange and Li active transport in human lymphoid cells U937 cultured in lithium media

Lithium transport across the cell membrane is interesting in the light of general cell physiology and becau- se of its alteration during numerous human diseases. The mechanism of Li4 transfer has been studied mainly in erythrocytes with a slow kinetics of ion exchange and therefore under the unbalanced ion distribution. Prolife- rating cultured cells with a rapid ion exchange have not been used practically in study of Li4 transport. In pre- sent paper, the kinetics of Li4 uptake and exit as well as its balanced distribution across the plasma membrane of U937 cells were studied at minimal external Li+ concentrations and after the whole replacement of external Na+ for Li+. It has been found that a steady state Li+ distribution is attained at a high rate similar to that for Na+ and Cl- and that Li+/Na+ discrimination under the balanced ion distribution at 1-10 mM external Li+ keeps on 3 and drops to 1 following blocking of the Na,K-ATPase pump by ouabain. About of 80% of the total Li+ flux across the plasma membrane under the balanced Li+ distribution at 5 mM external Li+ accounts for the equiva- lent Li+/Li+ exchange. The most part of the Li+ flux into the cell down the electrochemical gradient is a flux through channels and its small part may account for the NC and NKCC cotransport influxes. The downhill Li+ influxes are balanced by the uphill Li+ efflux involved in Li+/Na+ exchange. The Na+ flux involved in the countertransport with the Li+ accounts for about 0.5% of the total Na+ flux across the plasma membrane. The study of Li+ transport is an important approach to understand the mechanism of the equivalent Li+/Li+/Na+/Na+ exchange because no blockers of this mode of ion transfer are known and it cannot be revealed by electrophysiological methods. Cells treated with the medium where Na+ is replaced for Li+ are recommended as an object for studying cells without the Na,K-ATPase pump and with very low intracellular Na+ and K+ concentration.

Differential Effects of Polyphenols on Proliferation and Apoptosis in Human Myeloid and Lymphoid Leukemia Cell Lines

Background: Mortality rates for leukemia are high despite considerable improvements in treatment. Since polyphenols exert pro-apoptotic effects in solid tumors, our study investigated the effects of polyphenols in haematological malignancies. The effect of eight polyphenols (quercetin, chrysin, apigenin, emodin, aloe-emodin, rhein, cis-stilbene and trans-stilbene) were studied on cell proliferation, cell cycle and apoptosis in four lymphoid and four myeloid leukemic cells lines, together with normal haematopoietic control cells. Methods: Cellular proliferation was measured by CellTiter-Glo® luminescent assay; and cell cycle arrest was assessed using flow cytometry of propidium iodide stained cells. Apoptosis was investigated by caspase-3 activity assay using flow cytometry and apoptotic morphology was confirmed by Hoescht 33342 staining. Results: Emodin, quercetin, and cis-stilbene were the most effective polyphenols at decreasing cell viability (IC50 values of 5-22 µM, 8-33 µM, and 25-85 µM respectively) and inducing apoptosis (AP50 values (the concentration which 50% of cells undergo apoptosis) of 2-27 µM, 19-50 µM, and 8-50 µM respectively). Generally, lymphoid cell lines were more sensitive to polyphenol treatment compared to myeloid cell lines, however the most resistant myeloid (KG-1a and K562) cell lines were still found to respond to emodin and quercetin treatment at low micromolar levels. Non-tumor cells were less sensitive to all polyphenols compared to the leukemia cells. Conclusions: These findings suggest that polyphenols have anti-tumor activity against leukemia cells with differential effects. Importantly, the differential sensitivity of emodin, quercetin, and cis-stilbene between leukemia and normal cells suggests that polyphenols are potential therapeutic agents for leukemia.

Single-Cell Forensic Short Tandem Repeat Typing within Microfluidic Droplets

A short tandem repeat (STR) typing method is developed for forensic identification of individual cells. In our strategy, monodisperse 1.5 nL agarose-in-oil droplets are produced with a high frequency using a microfluidic droplet generator. Statistically dilute single cells, along with primer-functionalized microbeads, are randomly compartmentalized in the droplets. Massively parallel single-cell droplet polymerase chain reaction (PCR) is performed to transfer replicas of desired STR targets from the single-cell genomic DNA onto the coencapsulated microbeads. These DNA-conjugated beads are subsequently harvested and reamplified under statistically dilute conditions for conventional capillary electrophoresis (CE) STR fragment size analysis. The 9-plex STR profiles of single cells from both pure and mixed populations of GM09947 and GM09948 human lymphoid cells show that all alleles are correctly called and allelic drop-in/drop-out is not observed. The cell mixture study exhibits a good linear relationship between the observed and input cell ratios in the range of 1:1 to 10:1. Additionally, the STR profile of GM09947 cells could be deduced even in the presence of a high concentration of cell-free contaminating 9948 genomic DNA. Our method will be valuable for the STR analysis of samples containing mixtures of cells/DNA from multiple contributors and for low-concentration samples.

The Broken MLL Gene Is Frequently Located Outside the Inherent Chromosome Territory in Human Lymphoid Cells Treated with DNA Topoisomerase II Poison Etoposide

The mixed lineage leukaemia (MLL) gene is frequently rearranged in secondary leukaemias, in which it could fuse to a variety of different partners. Breakage in the MLL gene preferentially occurs within a ~8 kb region that possesses a strong DNA topoisomerase II cleavage site. It has been proposed that DNA topoisomerase II-mediated DNA cleavage within this and other regions triggers translocations that occur due to incorrect joining of broken DNA ends. To further clarify a possible mechanism for MLL rearrangements, we analysed the frequency of MLL cleavage in cells exposed to etoposide, a DNA topoisomerase II poison commonly used as an anticancer drug, and positioning of the broken 3’-end of the MLL gene in respect to inherent chromosomal territories. It was demonstrated that exposure of human Jurkat cells to etoposide resulted in frequent cleavage of MLL genes. Using MLL-specific break-apart probes we visualised cleaved MLL genes in ~17% of nuclei. Using confocal microscopy and 3D modelling, we demonstrated that in cells treated with etoposide and cultivated for 1 h under normal conditions, ~9% of the broken MLL alleles were present outside the chromosome 11 territory, whereas in both control cells and cells inspected immediately after etoposide treatment, virtually all MLL alleles were present within the chromosomal territory. The data are discussed in the framework of the “breakage first” model of juxtaposing translocation partners. We propose that in the course of repairing DNA topoisomerase II-mediated DNA lesions (removal of stalled DNA topoisomerase II complexes and non-homologous end joining), DNA ends acquire additional mobility, which allows the meeting and incorrect joining of translocation partners.