Characterization of fetal and adult B cell development and BCLL

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Characterization of fetal and adult B cell
development and B-CLL

• James Tung
• Nevada Cancer Institute

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Career to date

• B. A. in Genetics, University of California,
  Berkeley, 1987.
• Ph. D. in Genetics, Stanford University, 1997.
• Thesis advisors Drs. Leonard and Leonore
  Herzenberg
• Thesis Characterization of murine CD5 promoter
• Postdoctoral fellow 1997-2000. Stanford
  University.
• Advisor Dr. Samual Strober
• Project Involvement of B-1 cells in lupus-prone
  mice
• Research staff 2001-2007. Herzenberg Laboratory.
• Conduct research as a project leader. Supervise
  postdoctoral fellows and graduate students
• Research project Fetal vs. adult B cell
  development. Regulation of CD5 expression in
  developing T cells. Migration and replication of
  B-1 cells.

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Career to date

• Assistant Director, Stanford Shared FACS
  Facility 2003-2007.
• Director, Stanford Shared FACS Facility 2007.
• Director, Flow Cytometry Core, NVCI 2007-present
• Assistant Professor, NVCI 2008-present
• 1st year tenure track
• Main goals for moving to NVCI Research and
  Service.
• Improve flow cytometry infrastructure.
• Opportunity for collaboration in translational
  research.

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INBRE supported research

• Characterization of fetal and adult B cell
  development
• Development of a new imaging flow method to
  characterize B-CLLs and to identify cancer stem
  cells

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Fetal vs adult B cell development
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Background and significance
Fetal development Fetal hemoglobin
(?-globin) ?d T cells B-1 cells
Adult development adult hemoglobin
(ß-globin) aß T cells B-2 cells
Hematopoiesis differs between fetal and adult
stem cells. Implications for immune response and
disease progression
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Research Focus

• Study the activation profile between B-1 and B-2
  progenitors by intracellular phosphokinase
  staining in multi-parameter FACS analysis
• Define the genetic switch that controls fetal and
  adult B cell development in the mouse

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Research Focus

• Study the activation profile between B-1 and B-2
  progenitors by intracellular phosphokinase
  staining in multi-parameter FACS analysis
• Perform multiparameter flowcytometric analysis
  and intracellular phosphokinase/protein staining
  to distinguish potential signaling differences
  between B-1 and B-2 progenitors.
• These include
• Stat5 and JAK1/JAK2 in TSLP/IL-7 signaling
  pathway
• NFAT1/Calcineurin (needed in B-1 development)
• LEF1 and GSK3b in wnt signaling pathway
  (influenced by CD138)
• Brutons tyrosine kinase (Btk)

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Research Focus
Research Focus

• Define the genetic switch that controls fetal and
  adult B cell development in the mouse
• Sort B-1 and B-2 progenitors, isolate RNA from
  the sorted cells
• Perform Gene Array analysis with Affymetrix
  GeneChip Mouse Genome 430 2.0 Arrays
• Confirm observed differences with further studies

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Research Focus

• Development of a new imaging flow method to
  characterize B-CLLs and to identify cancer stem
  cells

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CLL

• Chronic lymphocytic leukemia (CLL) is the most
  common leukemia in western societies
• 30 of all leukemias.
• Incidence 2.7 per 100,000 in the USA
• gt90 of cases occur in patients gt50 years
• Higher incidence in men (MF2.8)

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Abnormalities in CLL

• Zeta-associated protein, 70 kDa, ZAP70
• Cytoplasmic tyrosine kinase normally expressed
  only by T cells and NK cells
• Variable expression within a given case cutoff
  for positivity 20
• Expression appears to be a stronger predictor for
  the need of early treatment than IgVH mutation
  status

Döhner et al NEJM, 2000
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Doublet formation and cell-cell communication
Sample 3 PercBPercC 120 Gating
scheme plot1_FITC-IgMa vs PE-IgMb Gate on PE
(R8) or FITCPE (R3) events Plot23_Doublet
gate Doublet events have large BF areas and low
aspect ratio (R4). Image gallery shows doublets
with or without CT AF647 accumulated at the
interface between the IgM cells
Green IgMa B cells from mouse 1 Orange IgMb B
cells from mouse 2 Red Cholera toxin subunit B
for lipid raft
James Tung, unpublished data
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Budget and Lab personnel

• Personnel 65
• Reagents and supply 35
• Lab personnel
• P. I. support 50
• One researach assistant 70 (Kristine Veys)

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INBRE is important for my career

• Allows me to continue my research on fetal and
  adult B progenitor cells
• Allows me to set up collaborations and begin
  interesting new researches
• Allows me to generate new data for future grant
  applications
• Allows me to improve Flow Cytometry work and
  education in Nevada

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Progress Update

• Obtain and breeding mice for neonate studies
  (months 1-6)
• Obtain fluorochrome conjugated antibodies and
  generate multicolor staining for sorting fetal
  and adult progenitor B cells (months 1-6)
• Sorting B-1 and B-2 progenitor cells for
• Gene expression arrays (bioinformatic core, UNR)
• Intracellular studies (month 6-12)
• Imaging flow (months 1-10), currently optimizing
  hybridization protocol for suspension cells.
  (Stanford blood center, Department of Pathology)

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Publications

• Published in 2008
• 1. Kerfoot, S.M., M. Szczepanik, J.W. Tung, and
  P.W. Askenase. 2008. Identification of initiator
  B cells, a novel subset of activation-induced
  deaminase-dependent B-1-like cells that mediate
  initiation of contact sensitivity. J Immunol
  1811717-1727.
• 2. Ghosn, E.E., Y. Yang, J. Tung, and L.A.
  Herzenberg. 2008. CD11b expression distinguishes
  sequential stages of peritoneal B-1 development.
  Proc Natl Acad Sci U S A 1055195-5200.

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Manuscript and Grant Application

• Manuscripts
• Tung, J., N. Mukhopadhyay, M. Phillips, L.A.
  Herzenberg, L.A. Herzenberg. B cell-B cell
  specific interaction involving B-1 cells
• Alipio, Z., Xu, D., Yang, J., Fink, L., Xu, W.,
  Ward, D., Tung, J., Ma, Y. Reprogrammed murine
  fibroblasts differentiated into hematopoietic
  progenitors are able to successfully engraft and
  repopulate in the bone marrow.
• Grant Applications
• Beckman Young Investigators Program, 300,000
  over 3 years.
• Melanoma Research Alliance Grant (collaboration),
  400-600k/year, 2-4 years.

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Collaborations

• Dr. Tracy George, M.D., Stanford University
  Pathology Department
• Imaging flow of CLL
• Dr. Karen Schlauch, Ph.D., Director, UNR Center
  for Bioinformatics. (INBRE supported Core)
• Gene Expression array analysis for fetal and
  adult B cell progenitors
• Dr. Douglas Redelman, Ph. D., Director, UNR
  Cytometry Center
• Flow cytometry education and training
• (INBRE supported)
• Dr. Wolfram Samlowski, M.D., Chief, section of
  melanoma, renal cancer, and immunotherapy, NVCI
• Melanoma stem cells and gene expression
• Dr. Louis Fink, M.D., Department of Laboratory
  Medicine, NVCI
• Prostate Cancer stem cells and biomarkers for
  detection
• Dr. Yupo Ma, M.D., Ph.D., Chief of
  Hematopathlogy, NVCI
• Induced Pluripotent stem cells in gene therapy

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Stanford University Dr. Leonard Herzenberg Dr.
Leonore Herzenberg Dr. David Parks Dr. Yang
Yang Dr. Eliver Ghosn Dr. Tracy George
NVCI Dr. David Ward Dr. Lou Fink Dr. Yupo Ma Dr.
Wolfram Samlowski
Nevada INBRE
Thank You
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