+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