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