Postdoctoral research studies in mouse T lymphocyte function - PowerPoint PPT Presentation

1 / 25
About This Presentation
Title:

Postdoctoral research studies in mouse T lymphocyte function

Description:

Identification of new mechanism of T cell migration. Industry experience at Promega in Cell-Based Assays ... actinomycin D (1mg/ml) Cyclohexamide (8mg/ml) ... – PowerPoint PPT presentation

Number of Views:30
Avg rating:3.0/5.0
Slides: 26
Provided by: Hea95
Category:

less

Transcript and Presenter's Notes

Title: Postdoctoral research studies in mouse T lymphocyte function


1
  • Post-doctoral research studies in mouse T
    lymphocyte function
  • utilizing gene expression arrays
  • Discovery of novel post-transcriptional
    regulation of
  • RANTES secretion
  • Identification of new mechanism of T cell
    migration
  • Industry experience at Promega in Cell-Based
    Assays
  • Developing turnkey reporter vectors for GPCR
    studies
  • Launching 1st mammalian cell line product
    supporting GPCR drug
  • screening portfolio

2
Discovery of novel post-transcriptional
regulation of RANTES secretion in memory T
lymphocytes
Immunity (2002)
3
Differentiation of Activated CD8 T Cells into
Memory T Cells
Death
resting, naïve CD8 T Cell
Survival and Differentiation
(IL-2Rblow CD44low)
activation, expansion, and differentiation
into effector cells
Memory CD8 T Cell
(IL-2Rbhigh CD44high)
4
Memory T cells contain high levels of RANTES mRNA
Average Difference Scores
Affymetrix U74V2 11k mouse gene array
5
T cell activation is required for secretion of
RANTES from memory CD8 T cells
memory
naive
20mg/ml anti-TCRb
50 ng/ml IL-15
2mg/ml anti-CD3
20 mg/ml anti-CD3
20mg/ml anti-CD3
20 mg/ml rat IgG
20mg/ml Rat IgG
1000
2000
3000
0
0
1000
2000
pg/ml RANTES
(pg/ml) RANTES
6
RANTES is secreted rapidly from CD8 memory T
cells after activation and does not require
transcription
2500
2500
memory
memory
2000
2000

1500
RANTES (pg/ml)
1500
IL-2 (pg/ml)
1000
1000
500
500
naïve
naïve
0
0
0
2
4
6
8
10
0
2
4
6
8
10
hours of stimulation
hours of stimulation
no inhibitor
actinomycin D (1mg/ml)
Cyclohexamide (8mg/ml)
Anisomycin (20mM)
hours of stimulation
7
RANTES mRNA is spliced and cytoplasmic in
resting memory CD8 T cells
NaïveMemory
Nuclear Cytoplasmic ratio of unspliced CD8 RNA
Cytoplasmic Nuclear ratio of processed RANTES
RNA
11
101
1001
Fold Increase in Relative Message Level
8
Immunohistochemical analysis of RANTES expression
in resting and activated CD8 memory T cells
18 hour activation
resting (ex vivo)
anti-RANTES
rat IgG
9
Conclusions 1
  • CD8 memory T cells store high levels of RANTES
    mRNA
  • which is used to rapidly produce RANTES
    protein after activation.
  • IL-15-mediated proliferation of CD8 memory T
    cells does not elicit RANTES
  • secretion, however, culture in IL-15 prior to
    T cell activation enhances
  • RANTES secretion after activation.
  • RANTES secretion after activation is rapid and
    not entirely dependent on
  • transcription, but is dependent on
    translation.
  • Intracellular RANTES protein is not detected in
    significant amounts prior
  • to activation.
  • RANTES mRNA is spliced and cytoplasmic in
    resting memory CD8 T cells
  • prior to activation.

10
Identification of a new mechanism of T cell
migration
Nature Immunology (2003) Nature Medicine (2004)
11
Trafficking of effector T cells to sites of
peripheral inflammation is not well understood.
  • More is known about migration and localization
    of
  • T cells to secondary lymphoid organs
  • Unfortunately, most infections and autoimmune
    disorders
  • dont occur in secondary lymphoid organs.
  • What directs T cells activated in lymph nodes
    to the peripheral
  • site of inflammation??

12
Memory T cell populations
TCM Lack immediate effector function CD62LhiCC
R7hi home preferentially to lymph nodes
TEM Immediate effector function CD62LloCCR7lo
home efficiently to non-lymphoid tissues
13
In vitro generation of effector and central
memory CD8 T cells
TEFF
IL-2
P14 TCR transgenic LN and SPL pulsed with LCMV
peptide (gp33-41)
Ficoll purify activated TCR transgenic T cells
7 days
2 days
IL-15
TCM
TEFF effector
TCM central memory
one P14 TCR transgenic mouse generates 108 TEFF
and TCM cells at 99 purity in 9 days
14
Regulation of mast cell degranulation
  • Pre-formed mediators (granules) histamine,
    proteoglycans, proteases, TNFa
  • 2. Lipid mediators e.g. leukotrienes,
    prostaglandins, PAF
  • 3. Cytokines Chemokines - e.g. IL-1, 4, 5, 8,
    13, 16, TNFa, MCP-1, RANTES, MIPa/b

15
Transwell Migration Assay
T cells
Mast cells or purified chemotactic agent
Mast cells sensitized with IgE, washed, and
stimulated with anti-IgE Abs. Mast cells/T cells
assembled in Transwell and placed at 37C for 3 h.
Cells in bottom well collected, T cell percentage
determined by FACS
of T cells that migrate to unactivated BMMC
given a migration index of 1
In some experiments, T cells in the bottom were
counted
16
Mast cells induce migration of TEFF but not TCM
cells
a
b

Untreated
4
10
TCM
IgE
TEFF
8
IgE anti-Ig
3
6
Migration index
Migration index
2
4
1
2
0
0
TEFF
TCM
100
101
10-1
0
102
CCL5 (nM)
plt0.0001
17
Enhanced BLT1 mRNA expression in TEFF cells
Signal values for select genes from Affymetrix
MG-U74 gene chip array.
TEFF
TCM
Real-time PCR analysis of BLT1 mRNA expression in
TEFF and TCM cells.
Granzyme D 2984 53 Granzyme E 2162 27 Granzyme
G 1048 20 Granzyme A 7444 184 BLT1 2578 169 CD62
L 3 836 CCR7 557 1665 CCR5 889 965 IL-2Rb 238
90 17506 CD44 298 251 b-actin 23398 24221
TCM
TEFF
BLTR1 threshold cycle 24.560.02 30.240.
09 b-actin threshold cycle
16.450.08 16.710.01 Fold increase in BLT1 mRNA
in TEFF cells normalized to b-actin
42.811.09
Threshold level settings used to obtain threshold
cycle values on ABI 7700 BLTR1 0.08, b-actin 0.1
r2 0.9042, calculated from signal values from
all probe sets comparing TCM versus TEFF cells.
18
Leukotriene biosynthesis
PLs
cPLA2
Arachidonic Acid
5-lipoxygenase (5-LOX) - (requires FLAP)
5-HPETE
5-LOX
MK-886
LTA4
LTA4 hydrolase
LTC4 synthase
LTB4
LTC4
19
Mast cell-induced migration of TEFF cells is
inhibited by the 5-LOX inhibitor MK-886
plt0.005 plt0.0001
20
Mast Cell/T Cell Migration Study Summary
  • Activated mast cells preferentially attract
    TEFF CD8T cells.
  • Gene expression array analysis identify BLT1
    chemotactic receptor as the
  • likely receptor conferring migratory activity.
  • Leukotriene synthesis inhibitors, BLT1 receptor
    antagonist, and LTA4
  • hydrolase-deficient mast cell studies verify
    BLT1 as receptor responsible
  • for migration.
  • TEFF, but not TCM CD8 T cells are required in
    mouse model of asthma

21
Improving Promegas luciferase reporter
technology portfolio
  • Strengths
  • pGL4 Best in class luciferase reporter
    technology
  • - codon optimization
  • - engineered backbone to reduce background
  • - Rapid Response luciferase reporter genes
  • Multiple luciferase reporters for multiplexing
  • - Firefly luciferase
  • - Renilla luciferase
  • - Click-beetle
  • Assay reagents to tailor to customers
    particular application
  • - brightness vs half-life of signal

22
Improving Promegas Luciferase reporter
technology portfolio
  • Addressable Weaknesses
  • Lack of turnkey assays
  • - Vectors
  • - Stable cell lines

23
Luciferase Reporter Vector Projects
  • Launch series of 6 vectors containing minimal
    promoter
  • - Used to study response element activity in
    the context of a weak promoter
  • - Vectors were demonstrated to have lower
    background and equivalent induced
  • luciferase expression compared to vectors
    using minimal TK promoter
  • Launch 2 turnkey vectors used to facilitate
    GPCR studies
  • - cAMP response element (CRE) and NF-AT RE
    luciferase reporter vectors

24
Stable Luciferase Reporter Cell Line Project
  • Lead scientist on team that launched
    GloResponse CRE and GloResponse
  • NF-AT RE stable HEK293 cell lines
  • These cell lines are the 1st mammalian cell
    line product for Promega
  • Launch process required interfacing with many
    operational divisions as well as
  • OEM cell line manufacturer.
  • Developed QA assay for OEM manufacturer
  • Authored Technical Manual

25
Post-Launch Support of Launched Vectors and Cell
Lines
  • Article in Cell Notes vol. 17 for vectors
  • Article in Cell Notes vol. 16 for cell lines
  • Technical services and sales training seminars
    and laboratory demonstrations
  • Meeting Presentations
  • Talk and poster at CHI GPCR Drug Discovery
    meeting 2006
  • Posters at MIPTEC and AACR in 2007
Write a Comment
User Comments (0)
About PowerShow.com