Immunosuppression via TCDD Activation of the Aryl

Report
Method Development in In Vitro Immune
Response
Jacob Huber
Dept. of Environmental and Molecular Toxicology,
Bioresource Research
Mentor: Dr. Nancy Kerkvliet
Overview
Background/Basis
Tools
Method Development
TCDD Experiments
Conclusions
Future Experiments
Background/Basis
What is the AhR?
A receptor modifying transcription

Affects immune system
Diverse ligands (stimulators) include products of
cellular metabolism:
-
Tryptophan, Arachiodonic Acid Metabolites
Tryptophan
www.acdlabs.com
Why is it important to understand how the
AhR works?
Could enable manipulation of immune system
Treatments for autoimmune diseases

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Type 1 Diabetes
Rheumatoid Arthritis
Others
Avoid negatives of current immunosuppresive drugs

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Often broadly suppress immune system
Targeted suppression
Maintain body‘s ability to fight disease
Most potent known ligand:

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
What is TCDD?
Persistant environmental
pollutant
Most toxic man made
chemical
Byproduct of combustion
Most potent agonist
(stimulator) of AhR due to:
-
High binding affinity
Resistance to Metabolism
Potent immune system
suppressor
2,3,7,8-tetrachlorodibenzo-p-dioxin
TCDD and Immunosuppresion
Normal (prolonged) immune response: T Cell
proliferation
TCDD induces CD4+ T Cells to differentiate into
CD25 expressing T Regulatory-like cells (Tregs)
Tregs inhibit T Cell proliferation, leading to immune
suppression
This TCDD induced change has been shown to be
AhR dependent in the Graft versus Host (GVH)
model
GVH Model
Models immune response
Donor T Cells transfered from a donor mouse to a host
These donor cells are alloreactive


Alloreactive: specifically reactive to non-self major
histocompatability complex molecules (MHC)
MHC molecules are expressed on the surface of cells
The donor and host mice are genetically identical,
except for polymorphims at the MHC
GVH Model - Response
Donor T Cells recognize
polymorphisms at the MHC of
the host (non-self)
They respond, proliferating
and differentiating into effector
cells
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Effector cells attack the host
they recognize as non-self
Leads to graft versus host
disease, resulting in organ
damage, death
Similar thing can happen
during bone marrow transplant
T Cell
TCDD and the GVH
In TCDD treated mice this immune response is
suppressed
T Cells do proliferate (recognize the host as non-self)
Differentiation is altered:

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T Cells differentiate into T Regs
Tregs prevent differentiation to effector cells
No effector cells, no attack mounted on host
No graft versus host disease
Proof of AhR Mediation
When AhR knockout mice (for donor T Cells) and TCDD are
used in GVH disease results

Indicates TCDD requires AhR
Suggests TCDD directly targets T Cells through the AhR
Previous experiments demonstrate:


AhR is needed in both CD4+ and CD8+ T Cells (T Cell subset)
CD8+ effects mediated by AhR competent CD4+
Question
Are T Cells directly targeted?
Possible that response is AhR dependent, but T Cells
are not the direct target
Which is the case?
Hypothesis
TCDD acts directly on CD4+ T Cells via the AhR to
alter differentiation and induce Treg activity.
Challenges:

Design an in vitro model to observe TCDD effects
Cheaper, Faster, Easier
No confounding variables

Use model to prove/disprove hypothesis
Model Design
Similar to in vivo GVH

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Dendritic cells (DCs) used in place of
host mouse
DCs cocultured with T Cells (CD4+
and CD8+) 3 Days
T cells will recognize DCs as non-self

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T cells in cultures without TCDD
should proliferate, express low levels
of CD25
With TCDD expected to proliferate,
exhibit higher CD25 levels
Mimic of GVH model
Dendritic Cell
Essential Tools
Fluorescent Labeled Antibodies
Cells are small!
Cells can be identified by
unique surface markers
Antibodies bind to surface
markers
Example:

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Dendritic Cells are
CD11c+, CD11bT Cells are CD3+, CD28+
faculty.ccbcmd.edu
Fluorescent Reagents
Ethidium Monoazide (EMA) penetrates dead cell
membranes and stains DNA


Can be used to determine viability:
Cell that it stains are dead
Carboxyfluoroscein Succinimidyl Ester (CFSE) can be
used to assess cell proliferation


Enters cells, undergoes cleavage by esterases, and is
distributed in the cytoplasm
Each time a cell divides, CFSE is distributed evenly to the
daughter cells
Beck Coulter Flow Cytometer
“Reads” fluorescent
reagents, labeled cells

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Lasers excite dyes
Dyes fluoresce specific
wavelengths (“channels”)
Can use 5 different
“colors” with our machine
AutoMACs Cell Sorter
Automated Magnetic Cell
Sorter
Allows isolation and
purification of cells based
on surface markers:

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Cell labeled with magnetic
antibodies
AutoMACS uses a magnet
to retain labeled cells
Positive and negative
populations are isolated
Method Development
(Isolation of Dendritic and T Cells)
Source of Cells?
Remember: T Cells and DCs
are cultured together, a source
is needed:
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Mice are used
T Cells are traditionally
isolated from spleens
DCs are isolated from
spleens, or bone marrow
1st Strategy: DC Isolation
Challenges:


Isolating DCs from bone marrow (BM) is time consuming (~9
days)
DCs are a rare population in the spleen (.75- 1.25% of cells)
Initially tried to isolate from BM, but it took too long
Decided to try using spleens instead
Splenic DC Isolation: 2nd Strategy
Use AutoMACs sorter
(described earlier)
DCs are CD11c+, CD11bLabeled cells with
corresponding magnetic beads
AutoMACs isolated CD11bcells, then CD11c+
Problems:


No Cells!
Expensive ($500+ for bead
kits)
Splenic DC Isolation: 3rd Strategy
Found a method in literature:

Digest Spleens in Collagenase
Collagenase is an enzyme that breaks peptide bond in collagen
Used to increase recovery

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Use a dense bovine serum albumin (BSA) gradient
Enrich by plastic adherence
Predicted yield 80%+ DCs
BSA Gradient
Based on density of Cells
Top of Gradient: RPMI-5
less dense than DCs
Bottom: Dense BSA,
greater density than DCs
Centrifuge 30 min/900g
Collect cells from the
interface (~30% DCs)
Enrichment by Plastic Adherence
DCs are transiently adherent to
plastic
After BSA Gradient, cells
cultured in a plastic dish:
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90 minutes in culture allows
adherence
Other (contaminating) cells
are washed off, DCs stick
Cells cultured overnight
After ~12 hours DCs become
non-adherent
Plates are washed and DCs
collected
Other cell types remain stuck
DC Isolation
Initial Problem: all cells dead at end!
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Collagenase was the culprit
Reduced time with collagenase to 30 min
End Results:
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DC go from ~.75% to greater than 80%
Process takes 2 days, better than 9 day BM isolation
T Cell Isolation
Much less of a challenge than
DC isolation!
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T Cells ~15% of splenic cells
A proven 1 day isolation
process exists
Method is simple, uses
AutoMACs
T Cell
AutoMACs T Cell Isolation
A Pan-T kit exists
specifically for T Cell
isolation
Cells from spleen are
labeled with CD11b,
CD45R, DX5, and Ter119
T Cell do not express any
of these markers
Negative cells are
selected
End Purity: >95%
DC:T Cell Ratio
Remember: DCs stimulate T Cell proliferation
The optimal T Cell:DC ratio must be determined:
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The best ratio will show clear division + increased CD25
expression
Cultured cells (wildtype) for 48 and 72 hours (based on
literature)
Tested 300:1, 60:1, 30:1 ratios (based on literature)
Labeled Cells with EMA, CFSE, CD25, CD4, and CD8
CD4 and CD8 mark T Cell subsets
Flow Cytometer
Results
Ratio Optimization
104
CD4 and CD 8 Gating
104
Viability
103
103
102
R3
101
101
CD8
EMA
102
R2
0
-101
-101
0
R1
0
200
400
600
FS
800
1000
(x 1000)
-101
0
101
102
CD4
103
104
Day 2 (48 hours) CD8
R7
R7
0
101
102
103
CFSE
3.3% Divided
17.8 MFI CFSE
75.3 MFI CD25
16.6% Viable
104
103
-101
0
101
CD25
102
103
CD25
-101
0
101
CD25
101
0
-101
-101
R7
1.2%
1%
102
103
.4%
102
30:1
104
104
60:1
104
300:1
-101
0
101
102
103
CFSE
3.8% Divided
19.1 MFI CFSE
87.6 MFI CD 25
13.5% Viable
104
-101
0
101
102
103
CFSE
3.4% Divided
15.3 MFI CFSE
93.7 MFI CD25
14.8% Viable
104
Day 2 CD4
104
30:1
104
60:1
104
300:1
R4
R4
-101
0
101
102
103
CFSE
2.5% Divided
25.7 MFI CFSE
94.2 MFI CD25
16.6% Viable
104
103
102
101
-101
0
101
0
-101
-101
0
101
.3%
2.2%
CD25
102
103
1%
CD25
CD25
102
103
R4
-101
0
101
102
CFSE
3.4% Divided
27.8 MFI CFSE
100.1 MFI CD25
13.5% Viable
103
104
-101
0
101
102
103
CFSE
3.9% Divided
25.3 MFI CFSE
98.6 MFI CD25
14.8% Viable
104
Day 3 (72 hours) CD8
R7
R7
0
101
102
103
CFSE
3.5% Divided
35.9 MFI CFSE
78.4 MFI CD25
12.3% Viable
104
103
8%
-101
0
101
CD25
102
103
102
-101
0
101
CD25
102
CD25
101
0
-101
-101
R7
6%
1.8%
103
30:1
104
60:1
104
104
300:1
-101
0
101
102
CFSE
7.9% Divided
58.6 MFI CFSE
59.9 MFI CD25
10.2% Viable
103
104
-101
0
101
102
103
CFSE
9% Divided
65.5 MFI CFSE
62.1 MFI CD25
9.1% Viable
104
Day 3 CD4
104
30:1
104
60:1
104
300:1
R4
R4
7.5%
103
6%
103
103
R4
-101
0
101
102
103
CFSE
3.8% Divided
54.3 MFI CFSE
76.9 MFI CD25
12.3% Viable
104
102
-101
0
101
CD25
102
CD25
101
0
-101
-101
0
101
CD25
102
2.4%
-101
0
101
102
CFSE
7.5% Divided
57.4 MFI CFSE
98.3 MFI CD25
10.2% Viable
103
104
-101
0
101
102
103
CFSE
8.4% Divided
55.9 MFI CFSE
141.7 MF CD 25
9.1% Viable
104
Best Ratio: 30:1
Day 3
104
104
Day 2
102
103
R7
101
0
-101
-101
0
101
CD8
CD25
CD25
102
103
R7
-101
0
101
102
103
104
-101
0
101
102
103
104
102
103
104
104
CFSE
104
CFSE
R4
103
102
101
0
-101
-101
0
101
CD25
CD4
CD25
102
103
R4
-101
0
101
102
CFSE
103
104
-101
0
101
CFSE
DC Ratio Conclusions
30:1 T Cell:DC ratio provides the most stimulation, CD25
expression
Day 3 (72 hours) exhibits the most distinct division,
highest CD25 expression
Splenic DCs are more potent than bone marrow derived
DCs
TCDD Experiment One
Use 30:1 Ratio to culture all cells
Culture AhR knockouts (-/-) with TCDD

Hope for proliferation, up-regulation of CD25
Culture AhR wildtypes (+/+) with TCDD:
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10-9,10-8 M
Hope to see some sort of dose response, less CD25
expression/proliferation
Culture AhR -/- and wildtype controls without
TCDD
Label + Flow
New Staining Scheme
CD4 and CD8 Gating
Stains:
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104
103
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102
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CFSE (T Cells)
EMA
CD4
CD25
CD62L (new)
CD4

R2
0
-101
CD62L=L-selectin
101
R1
-101
0
101
102
CFSE
103
104
Results – No Division!
Normal Division
104
104
No Division
103
103
R4
-101
-101
0
0
101
101
CD25
CD25
102
102
R5
-101
0
101
102
CFSE
103
104
-101
0
101
102
CFSE
103
104
Conclusions
Not enough DCs to assess purity

Used purity from previous experiment
No division:
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Not enough DCs?
Inadequate stimulation?
CD25/CD62L expression seen with division
Try it again
TCDD Experiment Two
10-8 M, 10-9M, 10-10M, and 10-11 M TCDD
Concentrations
Vehicle and no vehicle controls
16:1 ratio
Wildtype mice
Triplicate cultures
72 hours culture time
Label (same staining) + flow
Results - Viability
Viability
35
30
Percent Viable
25
20
15
10
5
0
1.00E-07 1.00E-08 1.00E-09 1.00E-10
Vehicle
Molar Concentration TCDD
No
Vehicle
Results – CD25 Expression
CD8s
800
800
700
700
600
600
500
500
MCF CD25
CD25 MCF
CD4s
400
400
300
300
200
200
100
100
0
0
1.00E-07
1.00E-08
1.00E-09
1.00E-10
Molar Concentration TCDD
Vehicle
No Vehicle
1.00E-07
1.00E-08
1.00E-09
1.00E-10
Molar Concentration TCDD
Vehicle
No Vehicle
Results – Dividing T Cells
Dividing CD4s
Dividing CD8s
350
104
102
300
100
98
MCF CFSE
MCF CFSE
250
200
150
96
94
92
90
88
100
86
84
50
82
0
1.00E-07 1.00E-08 1.00E-09 1.00E-10
1.00E-07
1.00E-08
1.00E-09
1.00E-10
Molar Concentration TCDD
Vehicle
Vehicle
No Vehicle
Molar Concentration TCDD
No
Vehicle
Results – CD62L Expression
CD8s (CD62L Downregulating)
2
5
1.8
4.5
1.6
4
1.4
CD8s Dividing%
CD4s Dividing %
CD4s (CD62L Downregulating)
1.2
1
0.8
3.5
3
2.5
2
0.6
1.5
0.4
1
0.2
0.5
0
0
1.00E-07
1.00E-08
1.00E-09
1.00E-10
Molar Concentration TCDD
Vehicle
No Vehicle
1.00E-07
1.00E-08
1.00E-09
1.00E-10
Molar Concentration TCDD
Vehicle
No Vehicle
Conclusions
TCDD does affects viability slightly
With greater TCDD concentration:
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CD25 increases (CD8s)
T cells divide to a greater extent
Percentage of cells downregulating CD62L
increases
Future Work
Future Work
TCDD experiment needs replication

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Validate results
Prove TCDD effects
Possible that TCDD acts on CD4+ T Cells , but effects
cannot be seen in this model:
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AhR could induce a primary change in T Cells
Many cell types are not present
Could require cell-cell orientations
Signals effecting Tregs could be diluted
An exhaustive in vitro study needs to be performed
Future Work
Model could screen for other AhR ligands
Strong AhR activators could have clinical applications

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Facilitate organ transplants
Treatment of autoimmune diseases
Current immunosuppression

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Global, passive
Cyclosporin A
Tregs are immunosuppressive
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Active
Avoid global suppression
Targeted suppression
Acknowledgements
Dr. Nancy Kerkvliet
Dr. David Farrer
Wanda Cranell
Linda Steppan
Danielle King
Everyone in the Nancy I. Kerkvliet Lab!
My family
References
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