+Effect of Inhibitory Cytokines on
PD1 & LAG3 Expression in Activated and Rested T Cells
Research SummaryAdeiyewunmi (Ade) Osinubi, Ruea Huang, Dr. Kunle Odunsi
Department of Gynecological Oncology, Roswell Park Center Institute, Buffalo New York, 14263
Roswell Park Summer Research Program
http://www.manifestdaily.com/vitamin-helps-turn-precancerous-cells-back-healthy-ones/
+Presentation Overview
IntroductionHypothesis/Experiment
Procedure/Method Results
Conclusion Other Experiments
+
Introduction
+ Multiple Co-Stimulatory Inhibitory Interactions Regulate T cell responses
PDL1 or PDL2CD80 or CD86
CD80 or CD86
HVEM
MHC I or II
PD1
CD28CTLA4ICOS
BTLA
TCRLAG3
+LAG3
Lymphocyte Activation Gene 3 Encoded by LAG 3 gene Immune checkpoint receptors Expressed on activated T cells, NK Cells, B Cells Diverse biologic effects on T Cells
+
Programmed Cell Death protein 1 Encoded by the PDCD1 gene Cell surface receptor Expressed on T cells and pro-B cells Immune checkpoint, down regulates immune system
Ligands PD-L1 & PD-L2
prevents T Cell activation
PD1
+Tumor Microenvironment Surrounding blood
vessels, immune cells, fibroblasts, signaling molecules, extracellular matrix
Effects the growth and evolution of cancer cells Hypoxia and extracellular
matrix role in metastasis
+Lag3 and PD-1 in Ovarian Cancer
Mastuzaki J, et. al. (2010) Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian caner. PNAS 107:7875-7880
CD8 TILs and TALs taken from patients show expression of both PD-1 and Lag3.
9
Ax700-AP
E-A
-102 103 104 105
-102102
103
104
105
96.12%0.00%
3.88%0.00%
Pacific Blue-A
PE
-A
-102 103 104 105
-102102
103
104
105 2.55% 1.97%
66.42% 29.07%
Ax700-A
Pac
ific
Blu
e-A
-102 103 104 105
-102102
103
104
105 0.00% 30.28%
0.00% 69.72%
CD8
PD
-1
LAG
-3CD8 PD-1
LAG
-3-102 103 104 105
-102102
103
104
105 0.00% 8.58%
0.00% 91.42%
-102 103 104 105
-102102
103
104
105
31.77%59.65%
7.53%1.05%
-102 103 104 105
-102102
103
104
105
56.24%0.00%
43.76%0.00%
CD8
PD
-1
LAG
-3
CD8 PD-1LA
G-3
30.3%
69.7%
3.9%
96.1%
2.0%
43.8%
56.2%
8.6%
56.2%
7.5%
1.9%
Coexpression of PD-1 and LAG-3 on CD8 TILs from murine ovarian cancer
+Tumor Microenvironment Cont…
Mastuzaki J, et. al. (2010) Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian caner. PNAS 107:7875-7880
+
Experiment
+
Hypothesis:TGFβ, IL-6, and IL-10 Cytokines will increase the expression of
LAG-3 and PD-1 in CD4 and CD8 T Cells
+ Procedure Isolate Splenocytes from C57 Black 6 Mice Grow Cells in 1640 Media for 72 hours in anti CD3 and
B7.1 coated plates During this time, treat the splenocytes with Cytokines
TGFβ, IL-6, IL-10
Harvest the Cells
Stain Cells with antibodies (FcBlock, CD8, CD4, PD1, LAG-3, CD-69)
Do Flowcytometry Assess and Analyze Data
+
Results
+
0 24 48 720
204060
CD4 expression over time
PD1 & LAG 3LAG 3PD1
hours expr
essi
on (%
CD
4 Ce
lls)
0 24 48 720
204060
CD8 cell expression over time
PD1 & LAG3LAG 3PD1
hours expr
essi
on (
%
CD8
Cells
)
6-12-15
LAG3 and PD1 expression over time
+
0 2.5 5 10 200
102030405060
CD8 cells treated with TGFβ expression
PD1LAG3 LAG3 & PD1
TGFβ (ng/ml)expr
essi
on (%
CD
8 Ce
lls)
0 50 100 2000
20406080
CD8 cells treated with IL6 expression
PD1LAG3 PD1 & LAG3
IL6 (ng/ml)expr
essi
on (
%
CD8
cells
)
0 2.5 5 10 200
102030405060
CD8 cells treated with IL10 expression
PD1LAG3PD1 & LAG3
IL10 (ng/ml)
expr
essi
on (
%CD
8 ce
lls)
6-18-15
Activated Cells with Cytokines
+
0 2.5 5 10 200
20
40
60
CD4 cells treated with TGFβ expression
PD1LAG 3LAG3 & PD1
TFGβ (ng/ml)
expr
essi
on (
%CD
4 Ce
lls)
0 50 100 2000
102030405060
CD4 cells treated with IL6 expression
PD1LAG3 LAG3 & PD1
IL6 (ng/ml)
expr
essi
on (
%CD
4 ce
lls)
0 2.5 5 10 200
102030405060
CD4 cells treated with IL10 expression
PD1LAG3PD1 & LAG3
IL10 expr
essi
on (
%CD
4 ce
lls)
6-18-15
+ Rested Cells
Activated cells for 72 hrs Rested Cells for 72 hrs Treated with three cytokines for 72 hrs Did not reactivate, Cells treated in non coated plates
+
0 2.5 100
5
10
15
CD8 Rested cells treated with TGFB
PD1LAG3PD1 & LAG3
TGFB Concentration
expr
essi
on (
%CD
8 ce
lls)
0 25 50 100 2000
5
10
15
CD8 Rested Cells treated with IL6
PD1LAG3PD1 & LAG3
IL6 Concentration
expr
essi
on (
%CD
8 ce
lls)
0 2.5 5 10 2002468
10
CD8 Rested Cells treated with IL10
PD1LAG3PD1 & LAG3
IL10 Concentrationexpr
essi
on (%
CD8
cells
)
6-18-15
0 2.5 5 10 20
+
0 2.5 1002468
10
CD4 Rested Cells treated with TGFB
PD1LAG3PD1 & LAG3
TGFB Concentration expr
essi
on (
%CD
4 ce
lls)
0 25 50 100 2000
0.51
1.52
2.5
CD4 rested cells treated with IL6
PD1LAG3PD1 & LAG3
IL6 Concentration
expr
essi
on (
%CD
4 ce
lls)
0 2.5 5 10 200
0.51
1.52
2.5
CD4 Rested Cells treated with IL10
PD1LAG3 PD1 & LAG3
IL10 Concentrationexpr
essi
on (%
CD4
cells
)
6-18-15
+ Reactivated Cells
Activated cells for 72 hrs Rested Cells for 72 hrs Treated with three cytokines for 72 hrs Reactivated the cells Cells treated in coated plates with lower concentration
of anti CD3 and B7.1
+
0
10
20
30
40
50
60
CD8 rested cells treated with var-ious combinations of cytokines
PD1LAG3 PD1 & LAG 3
expr
essi
on (%
CD8
cells
)
1 2 3 4 5 6 7 8TGFB - + - - + + - +
IL6 - - + - + - + +IL10 - - - + - + + +
+
0123456
CD4 rested cells treated with var-ious combinations of cytokines
PD1LAG 3PD1 & LAG3
expr
essi
on (%
CD4
cells
)
1 2 3 4 5 6 7 8TGFB - + - - + + - +
IL6 - - + - + - + +IL10 - - - + - + + +
+Conclusions
During activation, saw increase in LAG 3 in a concentration dependent manner Did not see huge effect on PD1 expression
In the Rested Cells, did not see quite similar trends, might be due to the fact that we did not reactivate
In the Reactivated Cells Single treatment of TGFB had greatest affect on LAG3
expression in CD8 cells Combination treatment increased PD1 expression In particular, combination of TGFB and IL10 had the
greatest affect
+ Treated Splenocytes with Glucose ELISA
Enzyme linked immunoabsorbent assay
Other Experiments
+ELISA for serum sLAG3
To test whether the level of soluble LAG3 protein in the serum samples from ovarian cancer patients correlates with clinical outcome.
Hypothesis: more sLAG3, less full length LAG3 is a good prognostic factor.
Method: ELISA
+Detailed Methods coat plate with capture antibody diluted in PBS, incubate overnight at
room temperature
wash 3x with Wash Buffer, block plate with Reagent wash 3x with Wash Buffer,
add 100 ul of sample/standard, incubate for 2 hours at room temperature
wash 3x with Wash Buffer, add 100 ul of detection antibody (biotin) diluted in Reagent Diluent,
wash 3x with Wash Buffer, add 100 ul Streptavidin-HRP solution
wash 3x with Wash Buffer, add 100 ul Substrate Solution (H2O2+TMB),
add 50 ul Stop Solution (2N H2SO4)
read plate at 450 nm (correct with 540 nm)
0 0.1 0.2 0.3 0.40
1000
2000
3000
4000
f(x) = 11250.6060841885 x^1.30971883367403R² = 0.99089193713876
Help to retrieve serum samples from the straws,
Work out condition using know concentration of sLAG3 protein,
Established Standard curve
Examined a few samples
+
Acknowledgments
+ Reference http://www.manifestdaily.com/vitamin-helps-turn-precancerous-cells-back-healthy-ones/
Mastuzaki J, et. al. (2010) Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian caner. PNAS 107:7875-7880
http://www.ncbi.nlm.nih.gov/pubmed/17932562
http://www.ncbi.nlm.nih.gov/pubmed/24955707
http://wirtzlab.johnshopkins.edu/research/tumor-microenvironment/
+
The End
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