Phosphatidylinositol 3-Kinase Mediates Bronchioalveolar Stem Cell Expansion in Mouse Models of Oncogenic K-ras-Induced Lung Cancer

Background: Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in Western countries. Developing more effective NSCLC therapeutics will require the elucidation of the genetic and biochemical bases for this disease. Bronchioalveolar stem cells (BASCs) are a putative cancer stem cell population in mouse models of oncogenic K-ras-induced lung adenocarcinoma, an histologic subtype of NSCLC. The signals activated by oncogenic K-ras that mediate BASC expansion have not been fully defined. Methodology/Principal Findings: We used genetic and pharmacologic approaches to modulate the activity of phosphatidylinositol 3-kinase (PI3K), a key mediator of oncogenic K-ras, in two genetic mouse models of lung adenocarcinoma. Oncogenic K-ras-induced BASC accumulation and tumor growth were blocked by treatment with a small molecule PI3K inhibitor and enhanced by inactivation of phosphatase and tensin homologue deleted from chromosome 10, a negative regulator of PI3K. Conclusions/Significance: We conclude that PI3K is a critical regulator of BASC expansion, supporting treatment strategies to target PI3K in NSCLC patients

PR1-Specific T Cells Are Associated with Unmaintained Cytogenetic Remission of Chronic Myelogenous Leukemia After Interferon Withdrawal

Interferon-alpha (IFN) induces complete cytogenetic remission (CCR) in 20-25% CML patients and in a small minority of patients; CCR persists after IFN is stopped. IFN induces CCR in part by increasing cytotoxic T lymphocytes (CTL) specific for PR1, the HLA-A2-restricted 9-mer peptide from proteinase 3 and neutrophil elastase, but it is unknown how CCR persists after IFN is stopped.We reasoned that PR1-CTL persist and mediate CML-specific immunity in patients that maintain CCR after IFN withdrawal. We found that PR1-CTL were increased in peripheral blood of 7/7 HLA-A2+ patients during unmaintained CCR from 3 to 88 months after IFN withdrawal, as compared to no detectable PR1-CTL in 2/2 IFN-treated CML patients not in CCR. Unprimed PR1-CTL secreted IFNgamma and were predominantly CD45RA+/-CD28+CCR7+CD57-, consistent with functional naïve and central memory (CM) T cells. Similarly, following stimulation, proliferation occurred predominantly in CM PR1-CTL, consistent with long-term immunity sustained by self-renewing CM T cells. PR1-CTL were functionally anergic in one patient 6 months prior to cytogenetic relapse at 26 months after IFN withdrawal, and in three relapsed patients PR1-CTL were undetectable but re-emerged 3-6 months after starting imatinib.These data support the hypothesis that IFN elicits CML-specific CM CTL that may contribute to continuous CCR after IFN withdrawal and suggest a role for T cell immune therapy with or without tyrosine kinase inhibitors as a strategy to prolong CR in CML

The Role of Antigen Cross-presentation From Leukemia Blasts on Immunity to the Leukemia-associated Antigen PR1

Cross-presentation is an important mechanism by which exogenous tumor antigens are presented to elicit immunity. Since neutrophil elastase (NE) and proteinase-3 (P3) expression is increased in myeloid leukemia, we investigated whether NE and P3 are cross-presented by dendritic cells (DC) and B-cells, and whether the NE and P3 source determines immune outcomes. We show that NE and P3 are elevated in leukemia patient serum and that levels correlate with remission status. We demonstrate cellular uptake of NE and P3 into lysosomes, ubiquitination and proteasome processing for cross-presentation. Using anti-PR1/HLA-A2 monoclonal antibody, we provide direct evidence that B-cells cross-present soluble and leukemia-associated NE and P3, while DCs cross-present only leukemia-associated NE and P3. Cross-presentation occurred at early time points but was not associated with DC or B-cell activation, suggesting that NE and P3 cross-presentation may favor tolerance. Furthermore, we show aberrant subcellular localization of NE and P3 in leukemia blasts to compartments that share common elements of the classical MHC class I antigen-presenting pathway, which may facilitate cross-presentation. Our data demonstrate distinct mechanisms for cross-presentation of soluble and cell-associated NE and P3, which may be valuable in understanding immunity to PR1 in leukemia

Priorities for synthesis research in ecology and environmental science

ACKNOWLEDGMENTS We thank the National Science Foundation grant #1940692 for financial support for this workshop, and the National Center for Ecological Analysis and Synthesis (NCEAS) and its staff for logistical support.Peer reviewedPublisher PD

Priorities for synthesis research in ecology and environmental science

ACKNOWLEDGMENTS We thank the National Science Foundation grant #1940692 for financial support for this workshop, and the National Center for Ecological Analysis and Synthesis (NCEAS) and its staff for logistical support.Peer reviewedPublisher PD

Isolation and characterization of a Chinese hamster ovary cell mutant with improved staining for indo‐1

Chinese hamster ovary (CHO) 10B2 cells do not stain well with indo‐1 and thus cannot be used for experiments to measure intracellular calcium using this dye. We have isolated a mutant CHO cell line (CHO IS1) that stains quite well with indo‐1 and that has virtually identical growth characteristics and heat sensitivity as the parent line. The mutant was isolated by sorting individual mutagenized cells with high indo‐1 fluorescence and cloning them. Since it has been reported that cells with multiple drug resistance (MDR+) can pump out various fluorescent dyes, the mutant and parent lines were characterized for Hoechst 33342 staining, Adriamycin toxicity, and P‐glycoprotein expression, which are markers of the MDR phenotype. P‐Glycoprotein was measured with the C219 antibody using flow cytometry. Multidrug‐resistant cells (CHRC5) were used as positive controls. The IS1 cells stained as well with Hoechst 33342 as fixed 10B2 cells, and much better than unfixed 10B2 cells. The IS1 cells were 10‐ to 30‐fold more sensitive to Adriamycin that the 10B2 cells, and both cell lines were much more sensitive than the CHRC5 cells. The amount of P‐glycoprotein was similar in both 10B2 and IS1 cell lines, but was about fivefold lower than the CHRC5 cells. Thus, the poor staining for indo‐1 in the 10B2 cells may not be caused by the P‐glycoprotein MDR pump, but by a different efflux pathway. Alternatively, the P‐glycoprotein may be altered and less efficient in the CHO IS1 cells. © 1994 Wiley‐Liss, Inc

Measurement of intracellular pH using flow cytometry with carboxy‐SNARF‐1

The new intracellular pH (pHi) dye carboxy‐seminaphthorhodafluor (SNARF‐1) was compared to the established dye 2,3‐dicyanohydroquinone (DCH) using flow cytometry. Both dyes give high‐resolution pHi measurements. SNARF‐1 remains trapped within cells much longer than DCH, so that pHi can be monitored during and after treatments with chemicals or hyperthermia. The toxicity of the dyes is similar, and both dyes can be used at concentrations that result in low toxicity to cells. Adequate staining of cells with SNARF‐1 is dependent on the cell concentration. The absolute pHi values indicated by SNARF‐1 are higher than values measured with DCH. However, the trends measured by both dyes are consistent, and both are useful for making pHi measurements. © 1993 Wiley‐Liss, Inc

Aberrantly Expressed Neutrophil Elastase (ELA2) Cleaves Cyclin E (CCNE) in the Nucleus and Cytoplasm of Acute Lymphocytic Leukemia Yielding Novel Leukemia-Associated Antigens

Abstract We have shown that donor-derived cytotoxic T lymphocytes (CTL) are specific for two HLA-A2-restricted peptides derived from CCNE (CCNE1144–152, ILLDWLMEV and CCNE2144–152, ILLDWLLEV) specifically lyse lymphoid and myeloid leukemia cells that aberrantly express CCNE. The CCNE protein is overexpressed in AML, ALL, and CML, and in solid tumors such as breast, lung, and gastric cancers. Furthermore, five low molecular weight forms (LMWFs) of CCNE, which are constitutively active to promote cell division, are found only in malignant cells, though it is not known how LMWFs are formed in leukemia. We hypothesized that the cleavage of CCNE into LMWFs occurs by enzymatic cleavage from aberrantly expressed ELA2 in leukemia, which renders the leukemia susceptible to killing by ELA2- and CCNE-specific CTL. Whole cell lysates from U937, HL60, and bone marrow from patients with B-ALL, but not from PBMC or bone marrow cells from healthy donors or ALL patients in remission, showed high expression of CCNE and LMWF by western blot (WB). Recombinant ELA2 added to B cell-derived whole cell lysates increased LMWFs, and the leukocyte elastase inhibitor Elafin prevented this cleavage. Subcellular fractions studied by coimmunoprecipitation showed that ELA2 was bound to CCNE in the nucleus, cytoplasm, and membrane-bound organelles of B-ALL, but not in normal cells. Because nuclear expression of CCNE increases during normal cell division, we studied healthy donor PBMC stimulated with anti-CD3 and anti-CD28 and found no expression of ELA2 or CCNE LMWFs by WB. We conclude that ELA2, normally expressed only in myeloid cells, is also expressed in some ALL blasts, and this data explains how CCNE LMWFs are formed in leukemia. Our findings also suggest how ELA2-mediated cleavage of the PML-RARα fusion product, required for leukemic transformation, could occur when ELA2 is aberrantly expressed in the nucleus. Finally, this work implies that overexpression of CCNE and the LMWFs that contain the immunogenic peptides could increase susceptibility of leukemia cells to CCNE-CTL lysis

Cytokine Production Signatures Are Intrinsically Associated with Human T Cell Maturation Stages

Abstract Our recent published studies have suggested that impaired immune reconstitution after allogeneic stem cell transplantation is associated with a greater proportion of circulating late memory T cells, defined phenotypically. To characterize the relationship between immunophenotypic markers of T cell maturation and functional attributes of T cells, we optimized an 8-color, 10-parameter cytokine flow cytometry (CFC) approach and studied T cells from healthy donors. T cells were exposed to stimuli that both bypass (PMA:Ionomycin, P:I) and signal through the T cell receptor (Staph enterotoxin B, SEB; and CMV pp65 peptide pools) and stained with CD45RA and CD27 to demarcate naïve (N, CD45RA+CD27+), and three progressively mature memory subsets: M1 (CD45RA−CD27+), M2 (CD45RA−CD27−), M3 (CD45RA+CD27−) CD4+ and CD8+ T cells. We assessed the 15 possible combinations of cells producing IL-2, IFNγ, TNFα, and MIP1β alone or in combination within maturation subsets. When we initially studied the production of individual cytokines, we found that the bulk of IL-2 production was produced by activated N and M1 cells in both CD4 and CD8 lineages. In contrast, IFNγ and MIP1β were produced by later maturation stages (M2 and M3) of CD4+ and CD8+ T cells. In contrast to the polarized production of individual cytokines at the extremes of the maturation spectrum, early and middle memory cells (M1 and M2) cells produced heterogeneous combinations of cytokines (e.g, IL-2+IFNγ+ and TNFα+MIP-1β+ cells). We also found that IL-2/IFNγ co-producing cells, shown to be particularly important for the control of chronic viral pathogens, exist mainly in the M1 and M2 stages, and not the M3 stage. The above results were consistent with both P:I and SEB stimulation, and across several healthy subjects tested. Our cross-sectional results were confirmed by in vitro differentiation experiments, wherein we sorted naive (CD45RA+CD27+) CD4+ and CD8+ T cells and demonstrated that their function evolved as expected following stimulation with PHA and IL-2, which resulted in differentiation into M1 and M2 cells in culture. Finally, we stimulated PBMC from healthy CMV-seropositive donors with a CMV pp65 peptide mixtures and examined maturation and cytokine production. Consistent with prior observations, most CMV-specific T cells were M2 and M3 cells. Surprisingly, the most abundant functional subsets consisted of cells producing either MIP1β alone or MIP1β and other cytokines. Consistent with our results following polyclonal stimulation, we found that IL-2/IFNγ co-producing CMV specific T cells existed in M1 and M2, but not in the M3 stage. These results demonstrate that: Functional cytokine signature is strongly associated with T cell maturation stage; Nearly all IL-2 production occurs in N, M1 and M2 cells; M3 cells produce little IL-2, but substantial amounts of MIP1β; IL-2/IFNγ co-production is rare in M3 cells, but exist in M1 and M2 cells, perhaps suggesting why late stage skewing of memory T cells may lead to functional T cell impairment in vivo; and that MIP1β is the most abundant cytokine produced by CMV-specific T cells. Overall, our results demonstrate that phenotypically defined maturation stages in both CD4+ and CD8+ T cell lineages are strongly associated with functional signatures irrespective of stimulus type, and that multidimensional analyses of human T cells may be beneficial when assessing human T cells in the clinical setting